Multivalent dengue virus vaccine

ABSTRACT

The present invention provides vaccine compositions of attenuated dengue virus. More specifically, the attenuated virus is produced by serial passage in PDK cells. The invention also provides methods for stimulating the immune system of an individual to induce protection against all four dengue virus serotypes by administration of attenuated dengue-1, dengue-2, dengue-3, and dengue-4 virus.

This application is a divisional application of U.S. application Ser.No. 09/535,117 filed on Mar. 24, 2000, now U.S. Pat. No. 6,638,514,issued Oct. 28, 2003, and further claims the benefit of priority under35 U.S.C. § 119(e) from U.S. application Ser. No. 60/126,313 filed onMar. 26, 1999, and U.S. application Ser. No. 60/181,724 filed on Feb.11, 2000.

INTRODUCTION

Dengue fever is caused by any of four serotypes of dengue virus,dengue-1, dengue-2, dengue-3, and dengue-4, which are transmitted tohumans by mosquitoes. In adults, dengue infections typically causeself-limited but incapacitating acute illness with fever, muscle pains,headache and an occasional rash. The illness may be complicated byhemorrhagic fever, which may be manifested by a positive tourniquettest, spontaneous petechiae, frank bleeding, and/or shock. Denguehemorrhagic fever is fatal in about 0.5% of cases. Patients who haveantibody from an earlier dengue infection who are subsequently infectedby another dengue strain have been shown to be at higher risk for denguehemorrhagic fever.

The mosquito vectors of dengue viruses are found in all tropical andsub-tropical areas of the world and in some temperate areas of theUnited States, Europe, Africa, and the Middle East. In recent years,endemic and epidemic dengue infections have occurred in Central andSouth America, Southeast Asia, India, Africa, the Caribbean and Pacificregions. Vector control is impractical.

An effective vaccine is needed which should confer protection againstall four serotypes of dengue.

SUMMARY OF THE INVENTION

The present invention satisfies the need discussed above. The presentinvention relates to vaccine compositions comprising attenuated denguevirus from all four serotypes. The attenuated virus is provided in anamount sufficient to induce an immune response in a human host, inconjunction with a physiologically acceptable vehicle and may optionallyinclude an adjuvant to enhance the immune response of the host.

Therefore, it is one object of the present invention to provide anattenuated dengue virus composition comprising attenuated more than onedengue virus selected from the group consisting of dengue-1, dengue-2,dengue-3, and dengue-4, in any combination.

It is another object of the present invention to provide methods forstimulating the immune system of an individual to induce protectionagainst dengue virus. These methods comprise administering to theindividual an immunologically sufficient amount of dengue virus from allfour serotypes which have been attenuated by serial passage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Occurrence of >Grade 1 symptoms as a result of vaccineadministration.

FIG. 2: Frequency of distribution of reactogenicity index by serotype.

FIG. 3: Table showing results of dose-ranging tetravalent dengue vaccinestudies.

FIG. 4: Table showing Immunogenicity of full-dose tetravalent denguevaccine in 10 subjects.

FIG. 5: Table showing details of selected formulations of tetravalentvaccine studies.

FIG. 6, A–H: Interferon γ production by PBMC collected from vaccinevolunteers and stimulated with serotype specific virus. All volunteersreceived only one serotype of vaccine. Graphs on the left (A–D) showresults from volunteers that were given the second dose around day 32.Graphs on the right (E–H) show results from volunteers that received thesecond dose around day 92. A response over 1000 pg/ml was seen justprior to the second dose in most volunteers. Only four volunteers had aresponse over 1000 pg/ml within the first 15 days of receiving the firstvaccine dose.

FIG. 7, A–D: Interferon γ production of PBMC collected from vaccinevolunteers receiving tetravalent vaccine. The PBMC were stimulatedindividually with each serotype of virus. Individual lines in each graphrepresent responses of one volunteer's PBMC to individual serotypes ofvirus. As with the monovalent vaccine recipients, late responses werenoted.

FIG. 8, A and B: Granzyme B mRNA production of PBMC collected frommonovalent and tetravalent vaccine volunteers. Cells were collected fromall individuals whose PBMC secreted ≧1000 pg IFNγ/ml at any time. Thisis a semiquantitative representation of the amount of mRNA detected byRTPCR. The upper chart (A) describes the intensity of bands seen for allsamples. The lower gel (B) is from selected volunteers to show examplesof positive and negative RTPCR assays.

DETAILED DESCRIPTION

The present invention provides attenuated dengue virus of all fourserotypes suitable for vaccine use in humans. The dengue virusesdescribed herein were produced by serial passaging of an infectiousdengue virus isolate in a suitable host cell line such as primary dogkidney cells so that mutations accumulate that confer attenuation on theisolate. Serial passaging refers to the infection of a cell line with avirus isolate, the recovery of the viral progeny from the host cells,and the subsequent infection of host cells with the viral progeny togenerate the next passage.

Preferably, the following attenuated viruses are used in thecompositions of the present invention even though other viruscompositions, of any of the serotypes, whether attenuated orinactivated, can be used in combination with the attenuated strainsdescribed in the present invention. The attenuated dengue-1 virus,derived from 45AZ5 isolate, PDK 20, was deposited on Apr. 30, 1999 underthe terms of the Budapest Treaty with the American Type CultureCollection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the accession number of VR-2648. Theattenuated dengue-1 virus, derived from 45AZ5 isolate, PDK 27, wasdeposited on Nov. 21, 2002 under the terms of the Budapest Treaty withthe American Type Culture Collection (ATCC) of 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A., and granted the accessionnumber of PTA-4810.

The attenuated dengue-2 virus derived from S16803 isolate, was depositedon Apr. 30, 1999 under the terms of the Budapest Treaty with theAmerican Type Culture Collection (ATCC) of 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A., and granted the accession number ofVR-2653.

The attenuated dengue-3 virus derived from CH53489 isolate, wasdeposited on Apr. 30, 1999 under the terms of the Budapest Treaty withthe American Type Culture Collection (ATCC) of 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A., and granted the accessionnumber of VR-2647.

The attenuated dengue-4 virus derived from the 341750 isolate, PDK 20,was deposited on Apr. 30, 1999 under the terms of the Budapest Treatywith the American Type Culture Collection (ATCC) of 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A., and granted the accessionnumber of VR-2652. The attenuated dengue-4 virus, derived from 341750isolate, PDK 6, was deposited on Nov. 21, 2002 under the terms of theBudapest Treaty with the American Type Culture Collection (ATCC) of10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., andgranted the accession number of PTA-4811.

The attenuated dengue-4 virus derived from the 341750 isolate, depositedunder the terms of the Budapest Treaty with the American Type CultureCollection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the accession number of VR-2652.

Serial passaging of a virulent (disease-causing) strain of dengueresults in the isolation of modified virus which may be attenuated,i.e., infectious, yet not capable of causing disease. These modifiedviruses are tested in monkeys for reduced infectivity. Those that havereduced infectivity are subsequently tested in humans. Humans are theonly primate that will exhibit signs of clinical disease. The virusesthat show minimal to no clinical reactivity but still infect and inducean immune response are attenuated.

In one embodiment of the invention, a virulent dengue isolate from allfour dengue serotypes was serially passaged in primary dog kidney (PDK)cells to derive the attenuated strains. Serial passaging was performedby infecting PDK cells with the virulent strain, incubating the infectedcells for several days, and collecting the supernatant culture fluidscontaining virus. The harvested virus was then applied to fresh PDKcells to generate the next passage.

Various passages in the series were tested for clinical effect afterfinal passage in fetal Rhesus monkey lung cells (FRhL). FRhL cells wereused to optimize virus titers wherein, in general, passage 1 wasconsidered the master seed, passage 2 was considered the productionseed, and passage 3 was considered the vaccine lot. Vaccines wereprepared at various PDK passage levels, and the vaccine products testedfor attenuation in monkeys and humans. The virulence of a passagedvirus, i.e., the ability to cause disease, was assessed by dailymonitoring of symptoms such as temperature (fever), headache, rash, toname a few. The passage was considered attenuated, as judged by theinability of this virus to elicit clinical signs of dengue disease invaccinees.

Propagation of the attenuated viruses of the invention may be in anumber of cell lines which allow for dengue virus growth. Dengue virusgrows in a variety of human and animal cells. Preferred cell lines forpropagation of attenuated dengue viruses for vaccine use includeDBS-FRhL-2, Vero cells and other monkey cells. Highest virus yields areusually achieved with heteroploid cell lines such as Vero cells. Cellsare typically inoculated at a multiplicity of infection ranging fromabout 0.01 to 0.005, and are cultivated under conditions permissive forreplication of the virus, e.g., at about 30–37° C. and for about 3–5days, or as long as necessary for virus to reach an adequate titer.Virus is removed from cell culture and separated from cellularcomponents, typically by well known clarification procedures, e.g.,centrifugation, and may be further purified as desired using procedureswell known to those skilled in the art.

The isolation of an attenuated virus may be followed by a sequenceanalysis of its genome to determine the basis for the attenuatedphenotype. This is accomplished by sequencing the viral DNA andidentifying nucleotide changes in the attenuated isolate relative to thegenomic sequence of a control virus. Therefore, the molecular changesthat confer attenuation on a virulent strain can be characterized.

One embodiment of the invention provided herein, includes theintroduction of sequence changes at any of the positions listed in thetable above, alone or in combination, in order to generate attenuatedvirus progeny. Viral genomes with such alterations can be produced byany standard recombinant DNA techniques known to those skilled in theart (Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates & Wiley Interscience, New York, 1989) forintroduction of nucleotide changes into cloned DNA. A genome may then beligated into an appropriate vector for transfection into host cells forthe production of viral progeny.

The ability to generate viral progeny through plasmid-mediatedintroduction of a viral genome can also be used to produce viruses withdefined molecular changes. In this embodiment of the invention, stablevirus stocks can be produced that contain altered sequences that conferdesired properties on the virus, for example, reduced virulence. Thisapproach can also be used to assess the effect of molecular changes onvarious properties of the virus, i.e. antigenic type, virulence, orattenuation by introducing desired sequence changes into the viralgenome, producing virus progeny from the genome, and recovering thevirus progeny for characterization. In addition, this approach can beused to construct a virus with heterologous sequences inserted into theviral genome that are concurrently delivered by the virus to generate animmune response against other diseases.

Construction of viral genomes with defined molecular changes can beaccomplished using standard techniques such as oligonucleotide-directed,linker-scanning or polymerase chain reaction-based mutagenesistechniques known to those skilled in the art (Zoller and Smith, 1984,DNA 3, 479–488; Botstein and Shortle, 1985, Science 229, 1193). Ligationof the genome into a suitable vector for transfer may be accomplishedthrough standard techniques known to those skilled in the art.Transfection of the vector into host cells for the production of viralprogeny may be done using any of the standard techniques such ascalcium-phosphate or DEAE-dextran mediated transfection,electroporation, protoplast fusion, and other techniques known to thoseskilled in the art (Sambrook et al., Molecular Cloning: A laboratoryManual, Cold Spring Harbor Laboratory Press, 1989).

For vaccine use, the attenuated viruses of the invention can be useddirectly in vaccine formulations, or lyophilized, preferably in astabilizer (Hoke, 1990, Am J Trop Med Hyg 43, 219–226), as desired,using lyophilization protocols well known to the artisan. Lyophilizedvirus will typically be maintained at about 4° C. When ready for use,the lyophilized virus is reconstituted in water, or if necessary, in astabilizing solution, e.g., saline or comprising Mg⁺⁺ and HEPES, with orwithout adjuvant, as further described below. All references citedherein are hereby incorporated in their entirety by reference thereto.

Thus, dengue virus vaccines of the invention contain as an activeingredient an immunogenically effective amount of more than oneattenuated dengue virus chosen from the group consisting of dengue-1,dengue-2, dengue-3, and dengue-4 as described herein. The attenuatedvirus composition may be introduced into a subject, particularly humans,with a physiologically acceptable vehicle and/or adjuvant. Usefulvehicles are well known in the art, and include, e.g., water, bufferedwater, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being rehydrated prior toadministration, as mentioned above. The compositions may containpharmaceutically acceptable auxilliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, and the like.

Administration of the live attenuated viruses disclosed herein may becarried out by any suitable means, including both parenteral injection(such as intraperitoneal, subcutaneous, or intramuscular injection), byin ovo injection in birds, orally and by topical application of thevirus (typically carried in the pharmaceutical formulation) to an airwaysurface. Topical application of the virus to an airway surface can becarried out by intranasal administration (e.g. by use of dropper, swab,or inhaler which deposits a pharmaceutical formulation intranasally).Topical application of the virus to an airway surface can also becarried out by inhalation administration, such as by creating respirableparticles of a pharmaceutical formulation (including both solidparticles and liquid particles) containing the virus as an aerosolsuspension, and then causing the subject to inhale the respirableparticles. Methods and apparatus for administering respirable particlesof pharmaceutical formulations are well known, and any conventionaltechnique can be employed. As a result of the vaccination the hostbecomes at least partially or completely immune to dengue virusinfection of the serotypes administered, or resistant to developingmoderate or severe dengue viral infection.

The vaccine composition containing the attenuated dengue viruses of theinvention are administered to a person susceptible to or otherwise atrisk of dengue virus infection to enhance the individuals own immuneresponse capabilities. Such an amount is defined to be a“immunogenically effective dose”. In this use, the precise amount againdepends on the subject's state of health and weight, the mode ofadministration, the nature of the formulation, etc., but generally rangefrom about 10² to 10⁶ pfu of each serotype of dengue virus per subject.The amount of virus vaccine of each serotype may be adjusted, i.e.increased or decreased, to result in a formulation which providessufficient protection from infection with the desired dengue virus. Whenthe four serotypes are combined, a preferred composition comprises equalamount of each dengue serotype. In any event, the vaccine formulationsshould provide a quantity of attenuated dengue virus of each of theserotypes sufficient to effectively protect the patient against seriousor life-threatening dengue virus infection of a serotype in the vaccineformulation, and possibly other serotypes if crossprotection occurs.

The attenuated dengue viruses of the invention of one particularserotype can be combined with attenuated viruses of other serotypes ofdengue virus to achieve protection against multiple dengue viruses.Typically the different modified viruses will be in admixture andadministered simultaneously, but may also be administered separately.

In some instances it may be desirable to combine the attenuated denguevirus vaccines of the invention with vaccines which induce protectiveresponses to other agents.

Single or multiple administration of the vaccine compositions of theinvention can be carried out. Multiple administration may be required toelicit sufficient levels of immunity. Levels of induced immunity can bemonitored by measuring amount of neutralizing secretory and serumantibodies, and dosages adjusted or vaccinations repeated as necessaryto maintain desired levels of protection.

The following examples are provided by way of illustration, notlimitation.

The following MATERIALS AND METHODS were used in the examples thatfollow.

Materials and Methods for Vaccine Production.

Virus strains. DEN viruses were passaged in primary dog kidney (PDK)cell cultures following isolation from human and mosquito sources. Table1 lists the strains that were adapted and passaged in PDK cells. Afterpassage in PDK cells, virus strains were further adapted to FRhL cellsfor seed and vaccine production. This consisted of an additional 3–4passages for final vaccine lot preparation. Parental virus strains, alsolisted in Table 1, were derived from low, cell culture passages in cellsthat were permissive for DEN virus replication.

Vaccine production. DEN vaccines for all four serotypes were prepared inFRhL cell culture using a similar procedure. FRhl cells, banked andpre-tested (see Table 2 for testing results) were removed from liquidnitrogen storage and plated in 150 cm² flasks in Eagle's minimumessential media (EMEM) (Biowhittaker, Waldersville, Md.) cell mediumsupplemented with non-essential amino acids, fetal bovine serum, FBS(2%) (Biowhittaker, Waldersville, Md.), and antibiotics. After theflasks reached confluency, medium was removed and flasks inoculated withDEN production seed diluted for an input of 0.01 MOI, and allowed toadsorb at 32° C. for 1 hr. Following adsorption and feeding with freshEMEM medium, flasks were returned to 32° C. for 4 days. On day 4post-inoculation, medium from all flasks was discarded and cellmonolayers were washed 3 times with 100 ml of Hanks BSS (Biowhittaker,Waldersville, Md.). After washing, flasks were fed with EMEM mediumcontaining 0.25% human serum albumin (HSA, Alpha Therapeutic Corp, LosAngeles, Calif.) replacing FBS. After an additional two days ofincubation at 32° C., supernatant culture fluids were removed from allflasks and pooled. After sampling for safety tests, the remainingculture fluids were pooled and clarified by filtration through a 0.45micron, non-protein binding membrane filter. The filtered fluids werepooled and mixed with an equal volume of stabilizer containing 15%lactose and 5% HSA. The bulk, stabilized fluids were stored at −70° C.until freeze-dried. For final vialing, bulk, stabilized fluids werethawed rapidly at 41° C. and aliquoted in 3 ml volumes in serum vials.Trays of vials were frozen to a temperature of −40° C. in a Hullfreeze-dryer, followed by drying for 1 day. Following capping, vialswere stored at −20° C. in a monitored freezer.

Vaccine testing. All cell banks used for virus preparations as well asseed and vaccine lots were tested for the presence of contaminatingagents. The test articles and results are listed in Table 2. Nodetectable contaminants were found in any of the products.

Rhesus monkey inoculation. Adult, male and female rhesus monkeys (6–15kg) were immunized with the DEN vaccine lots or parent viruses bysubcutaneous inoculation of 0.5 ml in the upper arm. Blood for virusisolation and antibody tests was drawn from the femoral vein prior toinoculation and every day for 14 days following inoculation. Blood wasalso drawn at 30 and 60 days following immunization. Virus challengeswere performed similarly.

Virus isolation by amplification in C6/36 cells. Virus isolation byC6/36 cell culture amplification has been described in Putnak et al,1996 (J. Infect Dis 174, 1176–1184). Briefly, following inoculation ofmonkeys, daily blood specimens were obtained from days 1 to 14. Serumwas separated and frozen at −80° C. For recovery of virus from sera,thawed sera were diluted 1:3 in cell culture medium and used toinoculate 25 cm² flasks containing monolayers of C6/36 mosquito cells.Following adsorption of virus, flasks were maintained at 28° C. in EMEMmaintenance medium. After 7 days, medium was changed and flasksincubated an additional 7 days. On day 14 post inoculation, supernatantculture fluids were decanted and frozen at −80° C. after mixing with anequal volume of heat-inactivated fetal bovine serum (FBS). Frozenspecimens were later assayed for infectious virus by plaque assay.

TABLE 1 Dengue virus strains used for development of live-attenuatedvaccines. Parental FRhL passages strain: passage Vaccine strain: passagefrom human PDK passages selected for seed and from Serotype OriginalIsolate isolate for vaccine prep vaccine prep human isolate DEN-1 Humanisolate, 20 × FRhL (with plaque 10, 20, 1: master seed 9 × FRhL (WestPac Nauru, 1974 selection and mutagenization 27 2: production 74; 45AZ5)with 5AZ); vaccine prep'd at seed p-20 caused dengue fever in 2 3:vaccine lot vols DEN-2 Human isolate, 1 × mosquito; 4 × PGMK 10, 20, 30,40, 1: master seed 4 × PGMK; (S16803) Thailand, 1974 50 2: production 2× C6/36 seed 3: vaccine lot DEN-3 Human isolate, 4 × PGMK; 5 × C6/36 10,20, 30 1: master seed 4 × PGMK (CH53489) Thailand, 1973 2: productionseed 3: vaccine lot DEN-4 Human isolate, 1 × mosquito 6, 10, 15, 20 1:pre-master 1 × mosq; (341750) Columbia, 1982 seed 5 × PGMK; (PDK-20only) 4 × FRhL 2: master seed 3: production seed 4: vaccine lot

TABLE 2 Pre-clinical testing of FRhl cell banks and DEN LAV seeds andvaccine lots. FRhl Vaccine cell Master Production Vaccine (Final Testbanks Seed Seed (Bulk) Container) Sterility x x x x x Mycoplasma x x xRT x x Hemadsorption x x x Cell culture safety x x (4 cell lines)Embryonated egg x safety Animal safety: adult x x mice Animal safety: xx suckling mice Animal safety: x x guinea pigs Animal safety: x rabbitsTumorgenicity x NA NA NA NA Karyology x NA NA NA NA Monkey safety: NA x(DEN-4) neurovirulence Monkey NA x infectivity/immuno- genicity Monkeyefficacy NA x (DEN-2, DEN-4) Infectivity (plaque NA x x x x assay)General safety NA x x Residual moisture NA x Reconstituted pH NA xReconstituted NA x osmolality Endotoxin NA x Identity (DEN) NA x x x

TABLE 3 DEN virus strain sets adapted to PDK cells, used for inoculationof rhesus monkeys. Den virus Inoc: PFU/ Mks viremic/Total Mkseroconverted/Total (GMT strain Viruses 0.5 ml (Mean days viremia)PRNT₅₀ at 1–2 mo post inoc) DEN-1, 45AZ5 PDK-0 3.3 × 10⁴ 4/4 (6.8) 4/4(760) (parent) PDK-10 7.0 × 10⁴ 4/4 (4.75) 4/4 (1030) (prod seed)*PDK-20 1.7 × 10⁴ 4/4 (4.5) 4/4 (640) (prod seed) PDK-27 1.8 × 10⁴ 0/4(0) 4/4 (50) (prod seed) DEN-2, S16803 PDK-0 5.0 × 10⁶ 4/4 (5) 4/4 (600)(parent) PDK-10 3.8 × 10⁵ 4/4 (4.75) 4/4 (570) (prod seed) PDK-20 2.2 ×10⁵ 4/4 (6.5) 4/4 (920) (prod seed) PDK-30 4.4 × 10⁵ 2/3 (3.3) 4/4 (640)(prod seed¹) PDK-30 2.1 × 10⁵ 3/3 (6.0) 3/3 (640) (prod seed²) PDK-401.0 × 10⁴ 2/4 (1) 3/4 (90) (prod seed) PDK-50 2.6 × 10⁶ 2/4 (1) 4/4(310) (prod seed¹) PDK-50 5.9 × 10⁵ 3/4 (3.25) 4/4 (280) (prod seed²)PDK-50   1 × 10⁶ ND 4/4 (270) (vaccine) DEN-3, CH53489 PDK-0 8.0 × 10³3/3 (3) 3/3 (660) (parent) PDK-10 2.5 × 10⁶@ 2/3 (1.3) 3/3 (150) (prodseed) PDK-20 1.0 × 10⁶@ 0/3 3/3 (130) (prod seed) PDK-30 9.3 × 10⁵@ 0/30/3 (<10) (prod seed) DEN-4, 341750 PDK-0 1.0 × 10³ 3/3 (4.7) 3/3 (420)(parent) PDK-6 1.7 × 10⁵ 1/4 (0.5) 4/4 (250) (prod seed) PDK-10 2.9 ×10⁵ 1/4 (1.3) 2/4 (90) (prod seed) PDK-15 5.5 × 10⁴ 1/4 (0.25) 2/4 (40)(prod seed) PDK-20 5.5 × 10⁴ 1/4 (0.25) 2/4 (70) (prod seed) PDK-20 1.2× 10⁵ 1/3 (0.3) 3/3 (50) (vaccine) 1, 2 Two separate monkey experimentalgroups. @Plaque assay performed in C6/36 cells.

Plaque assays. Infectious virus was titrated from amplified viremiaisolates or directly from monkey sera by plaque assay in Rhesus monkeykidney (LLC-Mk₂, ATCC CCL7) cells following the procedure ofSukhavachana et al. 1966 (Bull WHO 35, 65–66). Assays in C6/36 cells wasperformed as described in Putnak et al, 1996, supra.

Neutralization tests. DEN neutralizing antibodies were measured frommonkey sera using a plaque reduction neutralization test similar to thatused by Russell et al, 1967 (J Immunol 99, 285–290). Parent viruseslisted in Table 1 were used to measure the plaque reduction 50% endpoint(PRNT50) in serum specimens.

EXAMPLE 1

DEN virus modification in PDK cells and vaccine lot production. DENvirus strains selected for vaccine development had a variety of passagehistories prior to PDK passage. In the case of DEN-4 341750 there wasjust one mosquito passage before inoculation of PDK cell culture, whileDEN-1 West Pac 74 strain had a history of twenty FRhL cell passagesprior to PDK passage (Table 1). With the exception of DEN-3, all strainsadapted after a small number of PDK passages. For DEN-3, additionalefforts were required to increase viral input in early passages in orderto adapt this strain to PDK cells. As a general case after adaptation toPDK cells, DEN virus titers were found to be in the 10⁴–10⁵ PFU/mlrange. Attempts to increase titers were not successful and alternativecell substrates were sought for vaccine production. DBS-FRhL-2 (FRhL)cells were selected for this purpose for several reasons: 1) DEN virusesreplicate to titers of ca 10⁶ PFU/ml allowing manufacture of DENvaccines in these cells; 2) the cells have been used for the preparationof several DEN vaccines that have been tested in Phase I clinical trialswithout adverse reactions that may be related to the vaccine cellsubstrate; 3) FRhL cells are normal, rhesus monkey lung diploid cellsthat have no tumorigenic potential and are free of reverse transcriptaseactivity and contaminating agents; 4) since the cells are “normal”diploid cells there is no regulatory or other requirement to purify thevaccines; 5) FRhL cell banks can be established at cell generationsusable for vaccine manufacture starting with available, low passagecells. PDK passage therefore provides an excellent model for those whowish to study the empirical process of selective attenuation. But, justas PDK serial passage exerts a cumulative selection process, the furtherpassage in another cell substrate provides its own selective pressure.It is not known whether or not FRhL passage increases or decreases thevirulence of virus for humans. The use of stable cell lines that must befully characterized only one time is appealing. However, the publishedexperience with FRhL cells suggests that these cells may reverse ordestabilize biological properties acquired during serial passage in PDK(Halstead et al., 1984, Am J Trop Med Hyg 33, 654–665; Halstead et al.,1984, Am J Trop Med Hyg 33, 666–671; Halstead et al., 1984, Am J TropMed Hyg 33, 672–678; Halstead et al., 1984, Am J Trop Med Hyg 33,679–683; Eckels et al, 1984, Am J Trop Med Hyg 33, 679–683).

Adaptation of PDK-passaged viruses to FRhL was uniformly successful forall strains of DEN virus and was not dependent on PDK passage. Viraltiters from harvests of FRhL passages 1–4 ranged from 10⁵–10⁶ PFU/ml. Bythe third-fourth FRhL passage, vaccine lots of all of the DEN strain setviruses were prepared and tested as listed in Table 2. Data is alsoprovided in Table 2 for the FRhL cell bank testing as well as the masterand production seed testing. Results of these tests, required to ensurethe safety and the freedom from contamination, were negative, or fellwithin allowable specifications. For the DEN-4 341750 PDK-20 productionseed, monkey neurovirulence tests were performed. Results of this studycan be found in Hoke, 1990 (Am J Trop Med Hyg 43, 219–226). The DEN-4production seed as well as the DEN-4 parent virus that was used forcomparison were not neuropathogenic. Whether the remaining candidate DENvaccines need to be evaluated for neurovirulence remains questionablebased on data from this experience as well as other tests of DEN monkeyneurovirulence (personal communication).

EXAMPLE 2

Rhesus monkeys inoculated with PDK-passaged DEN viruses. The infectivityof DEN viruses passaged in PDK cells and designated as “strain sets” wascompared to parental, unmodified viruses for each serotype. Table 3lists the results of these studies where the degree of infectivity formonkeys was measured by the number of days of viremia that could befound in sequentially drawn serum two weeks following inoculation.Parental virus inoculation of monkeys resulted in 6.8, 5, 3, and 4.7mean days of viremia in groups of 3–4 monkeys inoculated with DEN-1,DEN-2, DEN-3, and DEN-4, respectively. For DEN-2 parent, additional data(not shown) has substantiated that infection with measurable viremia isvery reproducible over time using similar monkeys and isolationtechniques. Unfortunately, only partial data exists on viral titers inmonkey sera. Most of the data that exists comes from experience with theDEN-2 parent virus where monkey viremic blood was titrated in mosquitocell culture. Peak viral titers at 4–8 days post inoculation resulted intiters reaching 10⁵ PFU/ml of serum (Putnak et al, 1996, supra).

For each strain set, PDK passage results in modification of DEN virus asshown by reduced capacity of the virus to infect monkeys. For several ofthe strain sets this was clearly evidenced by the complete lack ofviremia at the highest PDK passage. Inoculation of monkeys with DEN-1 atPDK passage 27 resulted in 0 days of viremia in 4 monkeys. Thistranslates to 0 isolations out of a total of 56 bleedings tested. Asimilar result was found for DEN-3 PDK-20 and PDK-30. At PDK-30 for thisvirus, all evidence of monkey infectivity was lost, i.e., no viremia andno evidence of seroconversion in the monkeys inoculated with 10⁶ PFU ofvirus. The DEN-2 strain required the greatest number of PDK passages toattain modification of monkey infectivity. With this virus, at least 40passages in PDK cell culture were required for reduced viremia. Tocontrast this experience, the DEN-4 strain 341750 only required 6passages in PDK cells for a modified monkey infection. For another DEN-1strain, 1009, even after 50 PDK passages there was no evidence ofmodified monkey infection when compared to parental virus (data notshown). In conclusion, PDK cell passage appears to be an effectiveempirical method for modification and attenuation of various DENisolates. This is an unnatural host for DEN that probably placesselection pressure for virus populations that are suited for PDKreplication but not necessarily for replication in target cells inmonkeys and humans.

Materials and Methods for Candidate Vaccine Studies in Humans

Volunteers. Healthy male and female volunteers ages 18–45 were examinedand screened by a panel of tests, including blood chemistries,hematology, prothrombin time, partial thromboplastin time, urinalysis,rapid plasma reagin antibody, and serology for hepatitis B surfaceantigen and antibody to HIV. Volunteers were excluded on the basis ofpersistent significant abnormality or positive test. Female volunteerswere eligible to participate if they had a negative pregnancy testwithin 48 hours of vaccination and were willing to sign a consent formstating that they avoid conception using conventional contraception forthe 3 months following vaccination. In addition, volunteers wereexcluded if they had previous flavivirus immunity, which may affectresponses to dengue vaccines [Scott, 1983, J Infect Dis 148, 1055–1060]or a history of allergy to neomycin, streptomycin, or gentamycin. Priorflavivirus immunity was defined as having no detectable hemagglutinationinhibition antibodies (at a 1:10 serum dilution) against dengue types1–4, Japanese encephalitis, or yellow fever and no history of yellowfever vaccine or flavivirus infection.

Volunteers scored ≧70% on a written exam designed to test knowledge ofall aspects of the clinical trial. Informed consent was subsequentlyobtained from each volunteer in compliance with US 21 CFR Part50-Protection of Human Subjects. The clinical protocol conformed to allrelevant regulatory requirements, including the Declaration of Helsinki(Protocol), and Army Regulations 70-25-Use of Volunteers as Subjects ofResearch, and 40-7-Use of Investigational Drugs in Humans and the Use ofSchedule I Controlled Substances. The studies were approved by the HumanSubject Research Review Board, Office of the Surgeon General, U.S. Army,the WRAIR Human Use Research Committee, and the Institutional ReviewBoard, University of Maryland at Baltimore.

Study Vaccines. The study vaccines are listed in table 4. Vaccineviruses were passaged repeatedly in primary dog kidney cells and then infetal rhesus monkey lung (FRhL) continuous diploid cell culture as threeterminal passages to prepare seed and vaccine. Each candidate, beforetrial in volunteers, was confirmed to elicit substantially reducedviremia compared to its wild-type parent virus in vaccinated rhesusmonkeys. Adequate attenuation measured by infection of rhesus monkeysindicated that the dengue vaccine strains were appropriate vaccines forhuman testing.

Immediately before immunization, a vial of lyophilized vaccine wasreconstituted with sterile water for injection (USP). Afterimmunization, unused portions of rehydrated vaccine were maintained onice and titrated within 4 hours in LLC-MK₂ cell monolayers (Sukhavachanaet al. 1966, Bull WHO 35, 65–66). Each volunteer received between1.0×10⁵ and 4.5×10⁶ pfu of virus, depending on the candidate vaccineinjected (Table 4). The passage history of the individual study vaccinesis summarized below.

TABLE 4 WRAIR LIVE ATTENUATED DENGUE VACCINES Dose PDK Study Number of(×10⁵ Vaccine Passage* Year Site Volunteers pfu) Dengue 1 27 1991 CVD 104.4–45  (45AZ5) 20 1991 CVD # 10 7.7–38  1992 10 1991 CVD 9 2.8–3.5 19920 1984 USAMRIID 2 ? ## Dengue 2 50 1991 CVD 3 6.8 (S16803) 40 1996USAMRIID 3 5   30 1991 CVD 10 5.6–10  1992 Dengue 3 20 1992 CVD 61.0–1.4 (CH53489) 10 1992 CVD 3 3.8 0 1986 USAMRIID 2 ? Dengue 4 20 1989USAMRIID 8 1.0 (341750) 15 1991 CVD 3 4.8 TOTAL 10 — — 69 — *Primary dogkidney passage level # Center for Vaccine Development, University ofMaryland, Baltimore ## United States Army Medical Research Institute ofInfectious Diseases, Frederick, MD

Dengue 1 45AZ5 Vaccine: DEN-1 strain West Pac 74 was isolated from ahuman case of DEN fever on Nairu Island (Western Pacific) in 1974. Theisolate was passaged 20 times in FrhL cell culture and a vaccine lot wasprepared. Passages included mutagenization and plaque selection torecover a virus that was attenuated and suitable for human vaccination.Following vaccination of two human volunteers, the decision was made todiscontinue use of the vaccine due to DEN illness in one of thevolunteers. The vaccine was further attenuated by passage in PDK andFrhL cell cultures. The current, candidate vaccine is DEN-1 45AZ5PDK-20.

Dengue 2 S16803 Vaccine: The dengue 2 strain S16803 virus was derivedfrom a Thai virus isolate from a patient with dengue fever. The viruswas subjected to a total of 50 PDK passages, with terminal passage infetal rhesus monkey lung diploid cells (DBS-FRhL-2) for seed and vaccineproduction. Two vaccine candidates were initially prepared at the 30thand 50th PDK passage levels and selected for testing. Another vaccinecandidate was developed at the WRAIR from the same dengue 2 parentstrain S16803 virus and produced at the 40th passage level by the SalkInstitute (Swiftwater, Pa.).

Dengue Type-3 CH53489 Vaccine: Dengue type-3 strain CH53489 virus wasderived from a Thai strain, passaged 30 times in primary dog kidney(PDK) cells after initial passage in primary green monkey kidney (PGMK)and C6/36 insect cells. Virus from PDK passages 10, 20, and 30 was usedto inoculate fetal rhesus monkey lung diploid cell cultures.

Dengue 4 341750 Carib Vaccine: The dengue 4 vaccine candidate wasderived from a Caribbean strain of Dengue 4 (Columbia, 1982), passagedat the University of Hawaii, and manufactured at the WRAIR [Marchette,1990, Am J Trop Med Hyg 43, 212–218]. Antibody to the parent virusneutralizes other dengue 4 virus strains including H-241, the prototypestrain. Attenuation of the human isolate was achieved by passage 20times in primary canine kidney (PDK) cell cultures.

Study Design. A standard randomized, single-blind inpatient clinicalprotocol was used for all pilot studies. The majority of the studieswere conducted at the Center for Vaccine Development, University ofMaryland, Baltimore Md. The pilot studies of dengue 2 S16803 PDK 40vaccine and dengue 4 CH341750 PDK 20 vaccine were performed at theMedical Division, United States Army Medical Research Institute ofInfectious Diseases (USAMRIID), Ft Detrick, Md.

In the initial clinical studies of a vaccine, the highest availablepassage for a particular strain was tested first in three volunteers.Symptoms were monitored closely for three weeks, and if the volunteersremained well, the next lower passage was tested. If one or more of thevolunteers became ill, testing of lower passages of the vaccine strainwas not performed, as it was presumed lower passages were likely to beless attenuated. After testing of all acceptable passage levels in threevolunteers, the lowest level that did not cause illness was selected forfurther testing in up to seven additional volunteers.

To allow careful observation, prevent exposure to extraneous infectiousdiseases, and to prevent the possible infection of vector mosquitoes,volunteers were confined to the research ward from three days prior toinoculation until 20 days after immunization. All adverse experiencesoccurring within this period following administration of each vaccinewere recorded, irrespective of severity or whether or not they areconsidered vaccination-related. Acceptable safety of a vaccine wasdefined in advance as the absence of the following serious adverseevents: any severe clinical illness not explained by a diagnosisunrelated to the vaccination; persistent fever (oral temperature of≧38.5° C. for 4 determinations over 24 hours, a maximum daily oraltemperature of ≧38.5° C. on three successive days, or temperatureexceeds 40° C. on any individual determination); thrombocytopenia (fewerthan 100,000 platelets/mm³) or leukopenia (absolute neutrophil count<1000) on 2 consecutive determinations; or serum amino alaninetransferase (ALT) level of more than 4 times normal on 3 or moresuccessive days which is otherwise unexplained. In addition, anyexperience which would suggest any significant side effect that may beassociated with the use of the vaccine were documented as a seriousevent.

Volunteers were inoculated subcutaneously with 0.5 ml of undilutedvaccine on day 0. After immunization, vital signs were recorded every 6hours. The injection site was examined and the maximum diameter oferythema and induration measured and recorded daily. Clinical signs(fever [>37.8° C.], rash, vomiting, petechiae, and liver and splenicenlargement) and symptoms (malaise, headache, myalgia, arthralgia,nausea, and eye pain or photophobia) were assessed daily for the first20 days after immunization. Symptoms were graded as mild (noticedsymptom but continued ward activity) or severe (forced to bed bysymptom). If requested by the volunteer, painful symptoms were treatedwith propoxyphene hydrochloride; antipyretics were not used.Observations were recorded on a standard checklist of symptoms andphysical findings. Volunteers were discharged from the study ward on day21, and requested to return for serologic studies 1, 6, 12, and 24months after inoculation.

Two healthy flavivirus-immune volunteers were immunized at USAMRIID withthe parent strain of the dengue 1 45AZ5 vaccine and two years later withthe parent strain of the dengue 3 CH53489 vaccine. Medical records fromthe study were reviewed for presence or absence of the following signsand symptoms: fever, rash, malaise, headache, myalgia, arthralgia, andeye pain or photophobia. Viremia was measured daily. In contrast to thepresent trials, symptoms were not systematically recorded, and theintensity of symptoms was not graded. In addition, clinical experiencewith the dengue 4 341750 Carib PDK 20, given to 8 volunteers at USAMRIIDduring a later study, was extracted and summarized to compare with thoseof the current vaccinees [Hoke, 1990, supra].

Laboratory Evaluation. Blood was collected from volunteers every otherday and on day 31 for routinely available medical tests for hemoglobinand hematocrit, white blood cell count with differential count, plateletcount, and aspartate aminotransferase (AST) and alanine aminotransferase(ALT) levels. In addition, blood was collected every other day throughday 20 for virus isolation and antibody studies. Blood (20 ml) wasallowed to clot at 4° C. for ≦2 hours, sera was decanted into 1-mlaliquots, frozen and stored at −70° C. until study.

Virus Isolation. For determination of dengue viremia, serum was thawedand inoculated onto C6/36 mosquito cell monolayers and incubated at 28°C. for 14 days. Supernatant culture fluid harvests were assayed forvirus by plaque assay on LLC-MK₂ cells (Sukhavachana et al. 1966, BullWHO 35, 65–66). To quantitate the amount of virus in serum, a plaqueassay was performed on the C6/36 clone of Aedes albopictus mosquitocells [Hoke, 1990, supra]. Cell culture flasks were inoculated withdilutions of plasma and adsorbed at 35° C. for 1–2 hours. An overlaymedium consisting of Hank's Balanced Salt Solution and 0.75% agarose, 5%lactalbumin hydrolysate, 0.12 M NaHCO₃, and antibiotics was added andall flasks were incubated at 35° C. After 7 days, the flasks werestained with 5% liquid neutral red for 3–5 hours. Excess stain wasremoved and the plaques read after 18 hours.

Serology. Antibody tests included ELISA, HAI, and plaque reductionneutralization tests (PRNT) performed using a dengue virus of the sameserotype as the strain in the vaccine being tested. Detection ofanti-dengue IgM antibodies was performed by modification of an ELISA,where values >0.10 OD units were considered positive [Innis, 1989,supra]. The HAI test was performed by the standard technique modified tomicrovolumes using 4–8 units of individual antigens, using serumextracted with acetone to remove inhibitors [Clarke and Casals, 1958, AmJ Trop Med Hyg 7, 561–573]. PRNT assays were performed by the methoddescribed by Russell et al. [Russell, 1967, supra].

Statistical Analysis. The relationship between passage level and thefrequency and severity of reactogenicity was analyzed, for dengue 2vaccine S16803 (PDK 30, 40 and 50) and for dengue 3 vaccine CH53489 (PDK10 and 20), using the Cochran-Armitage test for trend and Spearman'scorrelations, respectively. The symptoms and signs independentlyanalyzed included the presence or absence, and the number of daysexperiencing eye symptoms, headache, malaise, myalgia, arthralgia, rashand fever (temperature >37.8° C.). The null hypothesis, that higher PDKlevel was not associated with lower reactogenicity, was evaluated at aprobability of five percent. By inspection of the data, the optimalpassage level for each virus was determined based on the clinical andimmunological responses of each volunteer. The passage level whichcaused no unacceptable side effects but which immunized about 80% ofvolunteers was selected for further development by the U.S. Army MedicalResearch and Development Command's Flavivirus Vaccine SteeringCommittee.

Definition of Infection by the Vaccine. Infection by vaccine is definedas replication of dengue virus in the volunteer, detected by presence ofserum type-specific neutralizing antibody or IgM anti-dengue antibodyafter immunization. Viremia was not included as necessary for diagnosisof infection as it was never detected in the absence of an antibodyresponse. A vaccine failure is defined as an unacceptable adverseclinical response or failure to develop convalescent IgM or PRNTantibodies.

EXAMPLE 3

Clinical Responses to Attenuated Dengue Vaccines

Dengue 2 S16803 Vaccine

The dengue 2 strain S16803 virus produced from the 50th passage in PDKcells was tested in three volunteers. The volunteers did well, with nooral temperatures >38.0° C. Two of 3 volunteers had transient mildsymptoms of malaise, headache, and eye symptoms (eye pain orphotophobia). Laboratory findings included mild ALT elevations (<2×normal) in 2 of 3, and mild leukopenia in 1 of 3 volunteers. Because ofthe acceptable safety profile of the PDK 50 vaccine, the next loweravailable passage, PDK 30, was selected for clinical evaluation.

The PDK 30 vaccine, tested in 10 subjects, was underattenuated andproduced symptoms compatible with mild to moderate dengue. Fourvolunteers (40%) developed low grade fever, to Tmax 38.5° C., over days9–14 post vaccination (median day 12). Eighty percent developed rash.The majority of volunteers experienced eye symptoms (10/10), headaches(9/10), and malaise (9/10), while 70 percent had ≧1 severe symptom ofheadache, eye pain and photophobia, malaise, or myalgia. Threevolunteers had mild elevation of their alanine aminotransferase (ALT), ameasure of liver pathology.

Because the PDK 30 vaccine was considered too reactogenic to testfurther in volunteers, the PDK 40 vaccine was produced from the masterseed. Two of three volunteers inoculated with PDK 40 developed a milddengue-like syndrome 9–10 days after vaccination, with low-gradetemperatures (<38.1° C.), rash, myalgias, and headache. Symptomsresolved spontaneously over several days without disability orrequirement for medication. Accompanying symptoms was an unanticipatedrise in serum liver enzymes, to a maximum ALT level of 199 IU/ml in one(4 times normal) and 77 IU/ml maximum ALT for the other (1.5 foldelevation from normal). The third volunteer remained asymptomatic butalso developed two-fold elevations in ALT (to max 10²). All laboratoryabnormalities resolved within days without intervention, and allvolunteers were discharged in good health 21 days after receipt of thevaccine. Because of the unusual frequency of hepatitis events associatedwith PDK 40 vaccine, no further development is planned for the product.

Table 5 summarizes the initial clinical experience with the WRAIR dengue2 vaccine. Decreased frequency of signs of fever and rash are apparentbetween passage level 30 and 50 vaccines. Furthermore, there is adecline in oral temperature from Tmax 38.5° C. towards normal withincreasing passage, but no change in duration of fever beyond one day.For the dengue 2 vaccine, the frequency and duration of eye symptoms,rash, headache, malaise and myalgia were significantly associated withpassage level.

TABLE 5 Clinical Responses in Recipients of Dengue 2 S16803 VirusVaccines days of Passage arthral- eye fever fever max Level malaiseheadache myalgia gia sx rash T > 37.8∞C (median) fever 2-S16803-30  9/10 9/10  7/10  4/10 10/10  8/10  4/10 9–14(12) 38.5 2-S16803-40 2/3 2/32/3 1/3 1/3 2/3 1/3 8.9 38.0 2-S16803-50 2/3 2/3 0/3 1/3 2/3 0/3 0/3 — —Symptom-days 2-S16803-30 2.2 3.6 2.4 1.7 3.3 5.4 0.5 2-S16803-40 2.0 1.72.0 1.0 5.7 1.7 0.7 2-S16803-50 0.6 0.7 0.0 0.3 1.0 0.0 0.0

TABLE 6 Clinical Responses in Recipients of Dengue 3 CH53489 VirusVaccines arthral- eye T > 37.8° C. max vaccine malaise headache myalgiagia sx rash (days) fever A: Number of patients having response3-CH53489-0 2/2 2/2 2/2 1/2 2/2 2/2 2/2 (5–9) 40.6 3-CH53489-10 1/3 2/32/3 1/3 1/3 2/3 1/3 (10,11) 38.2 3-CH53489-20 3/6 5/6 3/6 4/6 4/6 1/61/6 (3) 38.7 B: Symptom days 3-CH53489-0 3.5 4.0 4.5 2.0 3.5 7.5 5.03-CH53489-10 0.3 3.3 2.3 1.0 1.3 6.3 0.7 3-CH53489-20 1.7 2.8 1.0 2.01.3 0.8 0.2

TABLE 7 Viremia and Immune Responses to Dengue Vaccines Vaccine and daysof passage viremia range seroconversion level viremia (median) titer IgMHAI PRNT GMT31 GMT60 2-16803-30 10/10  6–12 (10)  3–1200  8/10 6/9 10/10343 262 2-16803-40 2/3 6–10 (8) NA 3/3 2/3 3/3 640 618 2-16803-50 0/3 —— 1/3 1/3 2/3 11 13 3-53489-0 2/2 3–10 (6) NA 2/2 2/2 2/2 2818 19953-53489-10 2/3 6–10 (8) 84–6600 1/3 3/3 3/3 710 153 3-53489-20 2/6  8–12(10) 12–138  2/6 1/6 3/6 556 4-341750-15 1/3 8–10 (9) 3–15  3/3 3/3 3/34-341750-20 5/8  8–14 (10) 10–1200 5/8 5/8 5/8 160

TABLE 8 Results of Phase I Trials of WRAIR Dengue Vaccine Candidates PDKMean Days Mean Illness Acceptable Number Number Range VaccinePassage^(a) viremia Score Reactogenicity Infected^(b) Seroconverted^(c)% Seroconversion Dengue 1 27 0.0 2.4 Yes 7 (70%) 4 (40%) 3–77 (45AZ5)[20] 1.0 3.6 Yes 10 (100%) 10 (100%) 10 5.0 3.9 Yes 7 (78%) 7 (78%)Dengue 2 [50] 0.0 5.0 Yes 2 (67%) 2 (67%) (S16803) 40 1.7 14.7 No  3(100%)  3 (100%) 30 2.2 19.1 No 10 (100%) 10 (100%) Dengue 3 [20] 0.611.0 Yes 3 (50%) 3 (50%) (CH53489) 10 2.3 15.3 No  3 (100%)  3 (100%)Dengue 4 [20] 3.8 6.6 Yes 5 (63%) 5 (63%) (341750) 15 0.6 20.7 No  3(100%)  3 (100%) ^(a)Primary dog kidney passage level ^(b)Defined asanti-dengue IgM positive or PRNT50 seroconversion ^(c)Defined as aneutralizing antibody titer > 1:10 (PRNT50) [ ] Strain proposed forexpanded clinical study

Dengue 3 CH53489 Vaccine. A dengue 3 vaccine (CH53489, PDK 0) developedat WRAIR was administered to two healthy yellow fever-immune malevolunteers as a 0.5 ml subcutaneous inoculation of 2×10⁴ pfu of virus.The immediate post immunization course was uneventful. By day 6, bothvolunteers were ill with moderately severe dengue fever characterized byhigh fever, chills, myalgias, headache, malaise, and a diffuseerythematous rash. Both volunteers developed thrombocytopenia andleukopenia but there were no signs of hemorrhagic fever. After a febrileperiod lasting five days, both men rapidly recovered and were well byday 21. Because of the severe illnesses experienced by both subjects, nofurther testing of this passage level was undertaken. Subsequently, PDK10 and PDK 20 passage levels were prepared as vaccine candidates.

The PDK 20 vaccine was given to 6 volunteers and resulted in mildreactogenicity. One subject experienced an early febrile illness on day3 with transient fever (Tmax 38.2° C.), pharyngitis, and cervicallymphadenopathy. No dengue virus was isolated from the volunteer'sserum. This subject was felt to have had an intercurrent illness withfever, which was not directly related to vaccination. Four out of 6volunteers developed short-lived mild dengue symptoms without rash;arthralgia, eye pain, and headache were the most frequent complaints.However, one volunteer had more severe symptoms of headache, malaise,and eye pain for three days. He also developed leukopenia and sustainedelevation in ALT levels; these laboratory abnormalities had resolved onfollow-up at day 31. Another volunteer had mild and reversible elevationof ALT alone, to less than 2× normal. Because the PDK 20 vaccine wassafe with marginally acceptable reactogenicity, the next lowestavailable passage vaccine virus (PDK 10) was tested.

The PDK 10 virus proved too reactogenic in recipients. One of threevolunteers developed low-grade fever on days 10 and 11 (Tmax 38.3° C.),and a florid rash for 13 days. Another volunteer developed persistentpruritus associated with waxing and waning hives on days 6 to 9 postvaccination, and tender cervical and axillary lymph nodes. Hesubsequently developed a maculopapular rash with malaise, headache, andmyalgia on days 10–12. This volunteer may have had an idiosyncraticallergic reaction to the vaccine, followed by a typical dengue-likeillness. These two volunteers also had laboratory abnormalities ofleukopenia and elevation of ALT levels to <2× normal, which resolved onfollowup on day 31.

Table 6 summarizes the response to dengue 3 CH53489 vaccines. Althoughthere was a trend for less frequent and shorter duration signs andsymptoms with passage, no passage reached statistical significance ineither analysis.

Dengue 4 341750 Vaccine. Eight volunteers received 10⁵ PFU of PDK 20vaccine [Hoke, 1990 supra]. Five volunteers developed a scarcelynoticeable macular, blanching rash and minimal temperature elevation(max 38.1° C.). Viremia and antibody response also developed in thesefive volunteers (63%).

A new DEN-4 341750 candidate vaccine was prepared from PDK passage 15,anticipating that the lower passage might be more infective. Threevolunteers received this vaccine and two experienced minimal symptoms.The third volunteer became ill abruptly on day 8 with fever, edematousswelling of the face and extremities, severe lassitude, rash, eye pain,photophobia, and arthralgias. Over the next three days, fever persistedwith Tmax of 39.6° C., but signs and symptoms resolved spontaneously.Because of this serious adverse reaction to vaccination, further use ofPDK-15 vaccine was terminated and PDK-20 was chosen for furtherevaluation.

EXAMPLE 4

Viremia and Immune Responses to Attenuated Dengue Vaccines

Table 7 describes viremia and immune responses with the WRAIR denguevaccines. The infectivity of the individual vaccines is summarizedbelow.

Dengue 2 S16803 Vaccine. No recipients of the PDK 50 vaccine developedviremia, yet two of 3 developed low-titer neutralizing antibody by day60. These findings suggested that the vaccine virus was diminished ininfectivity for humans. By contrast, two of 3 dengue 2 PDK 40 vaccineeshad demonstrable viremia, and all developed high titer antibody aftervaccination. As expected, infectivity of the dengue 2 PDK 30 vaccine washighest: viremia was detected in all 10 volunteers and all subjectsseroconverted with neutralizing antibody titers of >1:60 by day 60.

Dengue 3 CH53489 Vaccine. Dengue-3 virus retaining temperaturesensitivity and small plaque phenotype of the vaccine virus wasrecovered for 6 and 7 days in the 2 yellow fever immune recipients ofthe dengue 3 PDK 0 vaccine. Subsequently, high titered PRNT50 andhemagglutination inhibition (HAI) antibodies with asecondary-infection-like cross reactivity was measured in serumcollected on days 30 and 60 from both volunteers. Infectivity wassimilar in subjects who received the dengue 3 PDK 10 attenuated vaccine:2 of 3 developed viremia and vaccination induced neutralizing antibodiesin all. In contrast, 2 of 6 dengue 3 PDK 20 vaccinees had detectableviremia and three volunteers subsequently seroconverted, reflectingdiminished infectivity.

Dengue 4 341750 Vaccine. Eight volunteers received 10⁵ PFU of the PDK 20vaccine, and viremia and antibody response developed in five (63%). Thevaccine prepared from a lower passage of this candidate, PDK 15, wasmore infective. Virus was isolated from a single volunteer, on days 8and 10 following vaccination, with maximum titer of 15 pfu/ml. Thisvolunteer subsequently developed a neutralizing antibody titer of 450with a secondary HAI response, and was found to have been previouslyexposed to St. Louis encephalitis virus (PRNT titer 1:20 beforevaccination). The two volunteers without detectable viremia developedneutralizing titers of 1:10 and 1:40 by day 30 after vaccination.

EXAMPLE 5

Selection of Candidate Vaccines

The extended program of safety testing of the WRAIR PDK-attenuatedvaccines is shown in Table 8, which lists the salient features of thevaccines for each serotype. Increasing PDK passage resulted indecreasing mean illness score, which assesses duration and number ofsymptoms per volunteer. In addition, rising PDK passage was alsoassociated with decreased mean days of viremia, with the exception ofdengue 4 vaccines. Of the tested dengue 2, 3, and 4 vaccines, only onepassage level was judged safe and acceptably reactogenic, and suitablefor expanded clinical study: dengue 2 PDK 50, dengue 3 PDK 20, anddengue 4 PDK 20. However, the percentage of recipients infected declinedwith increasing PDK passage level. Seroconversion, defined as percentagewith neutralizing antibody titer ≧1:10 similarly declined within broadconfidence intervals.

Discussion

The WRAIR has longstanding involvement in the development oflive-attenuated dengue vaccines. Both the WRAIR and Mahidol denguevaccine programs have developed several live vaccines by attenuationthrough several passages (repeated growth in tissue culture) in dogkidney (PDK) cells. The results of the pilot testing in small numbers ofvolunteers established the safety of WRAIR candidate vaccines. Novolunteers among 65 recipients required emergent treatment of sustainedserious injury. Three volunteers suffered transient idiosyncraticreactions associated with dengue vaccination, resulting in withdrawal ofthe vaccines they received from further clinical development.Experimental infection with underattenuated vaccines, whileuncomfortable, was tolerable.

The clinical experience showed that increasing PDK passage of vaccineviruses increased attenuation for volunteers. This effect is best seenwith dengue 1 and dengue 3 viruses, where parental unpassaged virusesresulted in unmodified dengue fever and subsequent 20 PDK passagesacceptable reactogenicity. However, increasing PDK passage decreasedinfectivity of vaccine viruses, resulting in diminished immunogenicity.Furthermore, diminished viremia with vaccine viruses in humans appear tocorrelate with those in rhesus monkeys (with the exception of dengue 4PDK 15). These findings suggest that infectiousness of an attenuateddengue virus vaccine in volunteers proved equivalent to immunogenicity.

The relationship between passage level and reactogenicity should beinterpreted with caution, because subjects who experienced one symptomwere likely to experience several symptoms. As our analytic methodsassume independence of these symptoms, interpretations based onindependent p-values can be tenuous. Still, we believe rash showed astrong association with passage level (independent p=0.009 for presence,p=0.01 for duration). This is bolstered by a lack of significantcorrelation between rash and other symptoms, for either Dengue 2 or 3vaccine (Spearman's tests).

Only vaccines with acceptable safety profiles were selected for expandedclinical testing: dengue 1 45AZ5 PDK 20, dengue 2 S16803 PDK 50, dengue3 CH53489 PDK 20, and dengue 4 341750 PDK 20. Because of the broadconfidence intervals in seroconversion due to small numbers ofvolunteers, subsequent studies sought to increase the number ofrecipients of each of the four selected vaccines. In addition, furthertests will seek to determine whether immunogenicity of these attenuatedvaccines can be boosted through administration of two doses instead ofthe single dose used for these studies.

EXAMPLE 6

Expanded Study of Monovalent Vaccines; Monovalent Vaccines Given as TwoDoses; and Monovalent Vaccines Mixed as a Tetravalent Formulation Givenas One and Two Doses

Study Design: The objectives of these were to evaluate the safety andimmunogenicity of the four monovalent vaccines given as a single doseand then by two-dose vaccination schedules. Subsequently safety andimmunogenicity of the combination tetravalent vaccine were evaluated.Subjects were separately recruited from two sites, University ofMaryland at Baltimore and the WRAIR, Washington D.C. The first group of22 subjects were divided into 4 groups of 4 or 5 persons who eachreceived either a single dose of monovalent dengue or yellow fever 17Dvirus (Connaught). The 17D yellow fever vaccinees served as control andbenchmark for reactogenicity. Another 31 subjects were divided into 4groups of 7–8 persons who were given two doses of one monovalentvaccine, half at 1 month and the other half at 3 months. Finally 10volunteers were given 2 or 3 doses of the tetravalent vaccine. The first4 tetravalent recipients received vaccination at 0 and 1 month. Thelatter 6 tetravalent recipients were vaccinated at 0, 1 and 4 months.All subjects except the 10 tetravalent vaccine recipients were given avaccine serotype at random and in double-blinded fashion.

Subjects: Subjects were normal healthy adults age 18–50. All subjectswere seronegative for hepatitis B, C and HIV. All subjects wereseronegative for dengue 1–4, JE, SLE, and YF by hemagglutinationinhibition assay before entry into the study.

Vaccines: The four serotype vaccine candidates were originally isolatedfrom humans with clinical disease. Each were then modified by seriallypassage in primary dog kidney (PDK) and then fetal rhesus lung cells asdescribed above. These candidates were selected based on previous smallpilot studies in human volunteers. Each lyophilized monovalent vaccineswere reconstituted with sterile water and given in a volume of 0.5 cc.The doses of serotypes 1–4 were 10⁶, 10⁶, 10⁵ and 10⁵ pfu of Dengue 1,2, 3, and 4 respectively. The tetravalent vaccine dose was prepared bymixing 0.25 cc of each reconstituted monovalent and given in a finalvolume of 1 cc. The dose of the tetravalent vaccine was 1.1–2.8×10⁶ pfu.All vaccinations were given subcutaneously in the upper arm.

Clinical safety: Reactions to vaccinations were assessed by combinationof daily symptom diaries and periodic physician evaluations during the 3weeks after each vaccination. Subjects were housed in study quarters forclose observation for 5–7 days past the incubation period of 1 weekafter vaccination, a time period during which reactions and viremia weremost likely. Subjects were examined and queried specifically forsymptoms of feverishness, chills, headache, retroorbital pain, myalgia,arthralgia, rash and others. Each symptom was graded on a scale of 0(none), 1 (did not affect normal activity; did not require medications),2 (required medication or change in activity), or 3 (required bedrest orunrelieved by medication). The most common symptoms were grouped intofour categories. These categories were: 1) subjective fever and chills,2) headache and retroorbital pain, 3) myalgia and arthralgia and 4)gastrointestinal complaints which included nausea, vomiting andabdominal pain. A symptom index of each category was calculated by theproduct of the highest symptom grade for each day and the duration ofthe symptom expressed in days. If symptom occurred at all during 24hours it is assigned duration of 1 day. The Reactogenicity Index (RI) issimply the sum of the symptom indices for each category. The RIsummarized the vaccine reactions of each subject. The symptom categoryindices and RI allow for semi-quantitative comparison of vaccinereactions among subjects and vaccine serotypes.

Subjects were monitored for hematologic and liver toxicities by serialCBC, platelet counts, AST and ALT during the study.

Serious adverse events were defined as severe illness lacking otherlikely causes, fever >38.5° C. continuously for over 24 hours orTmax >38.5° C. for 3 consecutive days or a single oral temperature >104°C., neutropenia of <1,000/ml or thrombocytopenia of <90,000/ml on 2consecutive determinations, or serum ALT or AST >5 times normal.

Immunogenicity: Method of hemagglutination inhibition assay was done bymethod of Clarke and Cassals, 1958 (Am J Trop Hyg 7, 561–573) Dengue IgMand IgG were measured by capture ELISA in all but the last 6 tetravalentsubjects. Dengue and yellow fever neutralizing antibodies were measuredon Day 0 and 30 after each vaccination by plaque reductionneutralization test. The study endpoint determination was measurement ofany neutralizing antibody 30 days after last vaccination. Neutralizingantibody seroconversion is defined as 50% reduction in plaques atminimum of 1:5 serum dilution. Viremia was determined on sera from days7–14 after initial and second vaccination. Method used for virusisolation was a delayed plaque method adapted from Yuill, 1968 (Am JTrop Med Hyg 17, 441–448) using LLC-MK₂ or C6/36 cells for amplificationand Vero for plaque formation.

Data from the single-dose and two-dose studies were combined for thisreport. The subject characteristics are shown in Table 9. Total of fiftynine normal subjects were given dengue virus vaccines; forty ninereceived monovalent test articles and ten received tetravalent vaccine.Four received licensed 17D yellow fever vaccination (Connaught).

TABLE 9 Subject Characteristics No. Subjects (No. received Mean Vaccine2 doses) Sex Race Age Den-1 12 (8) 7M/5F 6W/6B 32 Den-2 12 (8) 7M/5F7W/5B 36 Den-3 13 (8) 9M/4F 8W/5B 36 Den-4 12 (7) 6M/6F 4W/6B/1H/1AmI 33Tetravalent  4 (4) 3M/1F 4W 26 YF 17D  4 (0) 3M/1F 3W/1B 30

EXAMPLE 7

Reactogenicity

Local reaction. Nineteen of 59 (32%) dengue vaccine recipients reportedmild arm pain at injection site. Of these 7 received DEN-1, 4 DEN-2, 1DEN-3, 1 DEN-4 and 5 received tetravalent. Only 5 reported any injectionsite pain after 24 hours. None affected use of the arm.

Systemic reactions. 20% of 59 dengue recipients reported no symptoms atall with their first vaccination while 70% of subjects were asymtomaticwith the second vaccination. The four subjects who received a third dosereported no symptoms associated with it. The most commonly reportedreactions from dengue vaccination were headache and myalgias. Theyoccurred in varying severity. FIG. 1 shows occurrence of > Grade 1symptoms from the first vaccination causing change in daily activitiesor taking of medications for relief. After the first dose of vaccine,five (8%) subjects, one serotype 1, one serotype 4, and threetetravalent, reported one severe grade 3 symptom of either chills,myalgia, headache or nausea for less than 1 day duration. No subjectsreported any grade 3 symptoms with revaccination.

The RI ranged from 0 to 35. Table 10 compares the reported reactogencityof each vaccine. The DEN-1 monovalent and tetravalent vaccines wereassociated with more reactogenicity. The second or third dose of alldengue vaccines uniformly caused few reactions, even in those subjectswith moderate to severe symptoms from the initial vaccination.

TABLE 2 Mean Reactogenicity Index Total Dose 1 Dose 2 RI Dose 3 VaccineSubjects RI (n) (n) RI (n) Den-1 12 7.4 (12) 0.5 (8) — Den-2 12 3.8 (12)0.3 (8) — Den-3 13 2.9 (13) 0.8 (8) — Den-4 12 3.7 (12) 0.5 (6) —Tetravalent 10 9.3 (10)  1.9 (10) 0.0 (4) YF 17D 4 3.8 (4)  — — — = notdone

FIG. 2 shows the frequency distribution of RI by serotype. Eightsubjects (14%) developed fever (>100.4° F.). Of the eight 4 receivedDEN-1, 1 DEN-2, 1 DEN-3 and 2 tetravalent. Highest and longest feveroccurred in a DEN-1 recipient with T_(max) of 103.3° F. and fever of 3days. Only one other subject, who also received DEN-1, had more than oneday of fever. Seven of the eight episodes of fever occurred followingthe first vaccination.

Sixteen subjects (27%) developed a generalized rash, involving the trunkand extremities from their first vaccination. Rash was usuallyerythematous, macular papular and mildly pruritic. Only 7 of the 16 withgeneralized rash had fever. Of the 16 subjects with rash five receivedDEN-1, two DEN-2, one DEN-3, three DEN-4 and five tetravalent. Rashtypically became noticeable by day 8–10 after vaccination and resolvedin 3 ?4 days. No subjects developed any petechiae, purpura or scarring.No subjects developed rash from revaccination.

Gastrointestinal symptoms were relatively common, occurring in a thirdof subjects, but they were mild and brief, lasting less than 24 hours.One DEN-4 recipient developed severe nausea associated with crampyabdominal pain for one day.

Six subjects (10%), 5 dengue and 1 yellow fever 17D recipient, developedtransient neutropenia with absolute neutrophil count less than 1000/ml.The lowest was 288 in a DEN-1 subject. Neutropenia typically resolves in2–3 days. No subject developed thrombocytopenia. There were noclinically significant elevations in AST or ALT.

As expected of this group of non-immune adult receiving their firstdengue virus exposure none developed any clinical evidence of denguehemorrhagic fever.

EXAMPLE 8

Immunogenicity

Viremia was detected in 10 subjects (17%), one received DEN-2, fourDEN-3, one DEN-4 and four tetravalent. No DEN-1 viremia was detected.The serotype(s) of the virus isolated from the tetravalent subjects haveyet to be identified. All detected viremias occurred after the firstdose of virus. Curiously fever occurred with viremia only in 3tetravalent recipients. All viremic subjects developed neutralizingantibody. One did not develop IgM or IgG response even with viremia.

Table 11 summarizes the antibody responses to monovalent vaccination.Neutralizing antibodies were detected more frequently than the IgM andIgG. No seroconversion was detected by IgM or IgG that was not alsofound by the PRNT₅₀ of 1:10 serum dilution. When present, IgM werepositive in 41% by 14 days after vaccination, in 17% by 21 days and 42%by 30 days. IgM typically peaked by day 30 after first vaccination. Asingle exception was in a tetravalent recipient whose IgM peaked threedays after his second vaccination. IgM can persist for more than 3months. The seroconversion rates by neutralizing antibody were 100%,92%, 54% and 58% for monovalent serotypes 1, 2, 3 and 4 respectively.When present, neutralizing antibody was typically detectable by day 30after first vaccination. No time points between day 0 and 30 wereassessed for neutralizing antibody. The second dose of vaccine boostedDEN-2 GMT by over four-fold, which was not seen with the otherserotypes. Two DEN-3 subjects seroconverted after a second dose ofvaccine, one at 1 month and the other at 3 months. They had notdeveloped neutralizing antibody after one dose. Interestingly theIgM/IgG patterns of these two subjects suggest a secondary responseafter their second dose suggesting they were immunologically sensitizedby the first dose.

Despite pre-entry negative hemagglutination inhibition assay for dengue,SLE, JE and YF 5 of 53 (9%) subjects tested developed a secondaryantibody response pattern with IgM to IgG ratio of <1.8. All 5 werenegative for homologous dengue neutralizing antibody prior tovaccination. This suggests a previous occult exposure to flavivirus. Wefound no significant difference between the mean RIs of secondary andprimary antibody responders (9.6 vs 5.8, p=0.19).

There were 12 monovalent subjects who did not develop IgM/IgG orneutralizing antibody. One received DEN-2, six DEN-3 and five DEN-4. Themean reactogenicity index for this group of antibody non-responders wasless than 1 which was significantly different from the mean RI of type2, 3 and 4 neutralizing antibody responders. (0.9 vs 4.9, p<0.003).

Our studies included 25 blacks and 31 Caucasian subjects. There was nosignificant difference between the mean RIs of these two racial groups.This is of interest because there is some epidemiologic evidencesuggesting milder dengue disease severity among blacks.

TABLE 11 Monovalent vaccine seroconversion rates by IgM and PRNT₅₀Seroconversions Seroconversions after 1^(st) dose by after 2^(nd) doseby Cumulative IgM (+) First dose IgM(+) Second dose Seroconversions byVaccine PRNT₅₀ GMT^(−1*) PRNT₅₀ GMT⁻¹ IgM PRNT₅₀ DEN-1 10/12   12/12(100%) 668 0/2 — 513 10/12   12/12 (100%) DEN-2 9/12 11/12 (92%) 112 0/30/1 559 9/12 11/12 (92%) DEN-3 4/13  6/13 (53%) 15 2/9 1/7 16 6/13  7/13(54%) DEN-4 5/12  7/12 (58%) 17 0/7 0/5 9 5/12  7/12 (58%) YF 17D 0/4  4/4 (100%) 2935 — — — 0/4   4/4 (100%) *used 30-day post vaccinationtiter; used value of 1 for negative titer in calculation − = not done

TABLE 12 Reactogenicity and Immunogenicity of Tetravalent VaccineRecipients Reactogenicity Vaccine Index Serotypes Neutralizing AbSchedule Dose Dose Dose Measured 30 days after Volunteer (months) 1 2 3Dose 1 Dose 2 Dose 3 33 0,1 16 0 — 1,2,3,4 1,2,3,4 — 34 0,1 0 0 — 2 1,2— 35 0,1 4 0 — 1,2,3,4 1,2,3,4 — 36 0,1 15 3 — 1 1,3 — 37 0,1,4 2 0 0 11 1,2,3 38 0,4 35 14 — 1,2 1,2,3 — 39 0,1,4 18 0 0 1,3,4 1,3 1,2,3,4 400,1,4 2 0 0 1 1 1,3 41 0,1,4 1 2 0 2 2 1,2,3,4 42 0,1 0 0 — 2 1,2 —

TABLE 13 Seroconversion rates of Monovalents and Multiple Doses ofTetravalent DEN-2 Vaccine DEN-1 Ab Ab DEN-3 Ab DEN-4 Ab Monovalent 1dose 12/12 11/12 6/13 7/12 Tetravalent 1 dose  7/10  6/10 3/10 3/10 p <.05 p > .07 p > .4 >.18 Tetetravalent 2 doses  9/10  6/10 5/10 2/10Tetetravalent 3 doses 4/4 3/4 4/4  2/4 

Age, Sex

Table 12 shows PRNT seroconversion results from the ten tetravalentvaccine subjects. The first 4 subjects received two vaccinations at 0and 1 month. One subject missed his second vaccination on day 30 and wasvaccinated on day 60. Six more subjects were to be vaccinated at 0 and 1month and if response was incomplete a third vaccination at 4 month wasadministered. Two subjects developed neutralizing antibody to all 4serotypes after a single dose. Another two tetravalent recipientsseroconverted to all 4 serotypes after vaccination at 4 months. Twoothers developed trivalent responses. A second dose of the tetravalentgiven at 1 or 2 months did not significantly increase seroconversions.The overall seroconversion rates in these 10 tetravalent subjects were100%, 80%, 80% and 40% for DEN-1, 2, 3 and 4 respectively.

EXAMPLE 9

A study was designed to evaluate interaction of each serotype componentin tetravalent vaccine by a 2-level 2⁴ factorial design.

Fifty-four subjects were given 15 permutations of 2 dose levels of eachserotype. Results are shown in FIG. 3. H, high dose, indicates undilutedvaccine, ranging between 10⁵–10⁵ pfu/ml; L, low dose, indicates a 1:30dilution of undiluted vaccine resulting in about 10^(3.5)–10^(4.5)pfu/ml.

Six subjects were given full-dose tetravalent vaccine at 0 and 1 month.If subject did not make tetravalent neutralizing antibody response, athird dose at 4 months was given. Results are shown in FIG. 4.

Four human subjects were given syringe-mixed full-dose tetravalentvaccine at time 0 and 1 month. Endpoints were clinical safety andneutralizing antibody at 1 month after second vaccination. T-cellresponses were measured in the first 4 subjects. Results are shown inFIG. 5.

Results indicate that tetravalent vaccine (16 formulations) were foundto be safe in 64 non-immune American volunteers. Reactogenicity varied.Four formulations elicited trivalent or tetravalent neutralizingantibody responses in all volunteers. In concordance with monovalentexperience, a second dose of tetravalent vaccine at 1 month did notinduce significant reactogenicity but also did not augment neutralizingantibody responses. End titration of neutralizing antibody responses isin progress. Memory interferon-gamma responses in T-cells can bemeasured in the absence of neutralizing antibody. Dosing intervals ≧4months may result in improved tetravalent seroconversion.

Discussion

These vaccines appear attenuated in humans when compared with historicaldescriptions of experimental infections with wild-type dengue. (Simmonset al., 1931, Manila Bureau of Printing) We used a numeric scale basedon self-reported symptom duration and severity to quantifyreactogenicity. Such method tends to over estimate vaccine-relatedreactions. Ideally it should be validated with cases of natural dengueinfection. However, imprecise the RI allowed us to reasonably comparesymptoms between individuals and groups. Results from testing themonovalent vaccines showed the degree of attenuation to be variableamong the four dengue vaccine candidates. 45AZ5 PDK20 is the leastattenuated, highest titer and resulted in uniform seroconversion. TheDEN-2 candidate, S16803 PDK50, similarly resulted in nearly 100%seroconversion with a benign reactogenicity profile. The Den-3 and Den-4had low reactogenicity profiles but seroconversion rates were only50–60%. It should be noted that the doses of type 3 and 4, the lessimmunogenic strains, are ten-fold less than that of types 1 and 2.

The second dose of virus was associated with remarkably littlereactions. However, the benefit of a second dose of monovalent vaccineat 1 or 3 month is small. Den-1 and 2 were already near uniformlyimmunogenic such that an additional dose may be superfluous.Nevertheless the GMT of Den-2 was boosted over four-fold. This may beevidence of low level viral replication after the second dose or thedose contains sufficient antigenic mass to elicit a booster response.This pattern of neutralizing antibody response has also been seen withsecond vaccination with 17D YF. (Wisseman, 1962, Am J Trop Med Hyg 11,570–575) The first dose of Den-3 may have sensitized the two monovalentsubjects who seroconverted after the second dose with secondary antibodyresponse pattern. This suggests that our neutralizing antibody assay maynot be sensitive enough to detect the appropriate immune response totype 3 vaccine candidates. The second dose did not add any newseroconverters to type 4. There was no obvious additional benefit ingiving a second dose of monovalent DEN-1 or DEN-4 with the dose andschedule tested.

Twelve monovalent subjects who did not make neutralizing antibodyresponse to monovalent vaccines also did not respond with measurabledengue IgM or IgG. All these non-responders received viable virus fromthe same vial that clearly replicated in other subjects. They developedno reactions to the vaccinations. Thus, by all indications there was noevidence of virus replication in these subjects. The mechanism for thisnonresponsiveness is unknown. It may be the result of lack of hostsubstrate necessary for infection or an effective innate immunity.

The value of multiple dosing may be more apparent in combinationlive-attenuated vaccines as a strategy to circumvent viral interference.Here dose of each component as well as the dosing interval may beimportant. Interference and enhancement can potentially occur whendengue viruses are given in combination. Four subjects developedneutralizing antibody to all 4 serotypes, two after the first dose, andtwo after a third dose at 4 months. Four of five volunteers who receivedrevaccination at 4 months seroconverted to 3 or more serotypes. Theexplanation of this difference may be that at one month aftervaccination there is sufficient cross-reactive neutralizing antibodiesto suppress replication of heterotypic viruses in the vaccine. Sabinfound that there was such transient cross protection lasting up to 3months when human subjects were given one serotype virus. (Sabin, 1959,Viral and Rickettsial Infections of Man. Philadelphia: JB LippincottCompany). Our future tetravalent studies will use a 0, 6 monthvaccination schedule.

The poor immunogenicity of DEN-3 and 4 may be that at 10⁵ pfu/ml Den-3and Den-4 doses are at replicative disadvantage compared to DEN-1 and 2,both of which are at 10⁶ pfu in the tetravalent formulation. We areexploring alternative production strategies to increase titers of DEN-3and DEN-4.

Without detecting viremia of all 4 viruses in the tetravalent respondersone cannot be certain that the presence of neutralizing antibodynecessarily imply replication of all 4 serotypes. Measured neutralizingantibodies may be cross reactive and of low avidity. This problem shouldbe addressed by looking at long-term persistence of antibody againsteach serotype. A sensitive and serotype-specific RT-PCR assay would beuseful to determine polyvalent viremia as evidence of viral replication.

Only two of the tetravalent vaccinees developed neutralizing antibody toall 4 serotypes after one vaccination. Such incomplete response totetravalent vaccine raises questions about risk of dengue hemorrhagicfever in the setting of exposure to virulent heterologous serotypes. Ifantibody-dependent enhancement is the pathophysiologic mechanism for DHFrisk may be present even when all four serotype antibodies are elicitedby vaccination but one or more serotype antibody wanes differentiallybelow neutralizing threshold. We report below that TH1 T-cell responsecan be measured in these tetravalent vaccinees even in the absence ofneutralizing antibody. Would that be sufficient to protect? Thesequestions may only be answered by careful long-term field testing oftetravalent vaccines in endemic areas.

In conclusion, our results indicate that the four serotypes are variablyreactogenic as monovalent vaccines with type 1 more so than serotypes 2,3 and 4. Serotypes 1 and 2 elicited neutralizing antibody in >90% whileserotypes 3 and 4 are less immunogenic. The tetravalent combination issafe, reasonably well-tolerated and induced neutralizing antibody to all4 serotypes in four of ten subjects. Two doses of tetravalent vaccinedid not improve seroconversion rates at the one or two-month dosingintervals tested. A longer dosing interval of over 4 months may improveseroconversion rate.

EXAMPLE 10

Material and Methods for T-Cell Response to Dengue Vaccines

Subjects. Thirty-five healthy adult volunteers ages 18–50 (21 males, 14females) participated in a phase I clinical trial, conducted by theWalter Reed Army Institute of Research, involving candidate dengue virusvaccines. The participants were selected from a group of volunteersbased upon the absence of circulating anti-flavivirus antibody.Additional selection criteria was HIV negative status and good healthbased upon a physical exam and responses to a questionnaire.

Vaccine groups. Thirty individuals randomly received two doses of a liveattenuated monovalent vaccine; four received two doses of a liveattenuated tetravalent vaccine. One monovalent recipient (volunteerID 1) quit the study after only receiving the first dose. Prior tovaccination, there was no detectable hemagglutination-inhibiting serumantibody to dengue virus types 1–4, Japanese encephalitis virus, St.Louis encephalitis virus, or yellow fever virus in any of thevolunteers. Each dose was given as a 0.5 ml subcutaneous injection ofundiluted virus(es).

PBMC collection. Peripheral blood (8 ml) was collected from eachvolunteer by venipuncture into Vacutainer Cell Preparation Tubes (CPT)[Becton-Dickinson, Franklin Lakes, N.J.] on day 0 and at five timepoints after the first dose but prior to the second dose (days 3, 7, 9,14, 28/ 30/ 31/ 60 or 91). Blood was also collected on the day of thesecond dose and at four time points afterwards (days 3, 7, 9 and 14 postsecond dose). The time of administration of the second dose, dependingon the volunteer, thus was approximately 1–3 months after the firstdose. Variation in collection times around 1 month occurred due tovariation in volunteer scheduling. Cells were separated from whole bloodby centrifugation at 1000×g for 30 minutes. PBMC were collected (thecell layer above the gel in the CPT tube) and washed twice in Hank'sbalanced salt solution (Life Technologies, Rockville, Md.) withcentrifugations at 500×g. Isolated PBMC were resuspended in 4 ml (perCPT tube) of Cell Freezing Media/DMSO (Sigma, St. Louis, Mo.) and frozenin 1 ml aliquots overnight at −70° C. The PBMC were then transferred tovapor phase liquid nitrogen for long term storage.

Vaccine viruses. The following live attenuated dengue virus strainsdescribed above were used in the monovalent vaccines: 45AZ5PDK20 (DEN1), S16803PDK50 (DEN 2), CH53489 (DEN 3), 341750PDK20 (DEN 4). Thetetravalent vaccine was an equal mixture of all four of these strains.

Cell culture viruses. The following dengue viruses, propagated in Verocells, were used for PBMC stimulation in culture: Westpac 74 (DEN 1),S16803 (DEN 2), CH53489 (DEN 3), and TVP360 (DEN 4). All four serotypeswere provided by Dr. Robert Putnak in 1 ml aliquots and stored at −70°C. until use. The virus titers ranged from 0.30–2.4×10 6 pfu/ml.

Bulk culture of PBMC and stimulation with live virus. Frozen vials ofPBMC were removed from liquid nitrogen storage and gently thawed at 37°C. PBMC were washed twice with RPMI medium 1640 (Life Technologies,Rockville, Md.) and suspended in complete media containing 10% humanmale AB serum (Sigma) plus supplements [penicillin (100U/ml)-streptomycin (0.1 mg/ml)-fungisone (0.25 mg/ml) [Sigma], 2 mML-glutamine (Life Technologies), and 0.5 mM 2-mercaptoethanol (Sigma)].The cells were suspended at a concentration of 2.5 million cells/ml.Some assays required 3.25 million cells/ml. The PBMC (100 ml) were addedto individual wells of a 96-well V-bottom plate (Costar, Acton, Mass.).An equal volume of dengue virus 1, 2, 3, or 4 diluted in 10% completemedia at a concentration of 3000 to 24000 pfu/100 ml was added to eachwell. Control wells received an equal volume of medium without virus.The cells were then cultured at 37° C. in 5% CO₂ for four days.

Immunoassay.

A chemiluminescent immunoassay was done to determine the quantity oflymphokine secreted in tissue culture supernatant at the end of 4 daysof culture. A 96 well immunoassay plate, Microlite 2 (DynatechLaboratories, Inc., Chantilly, Va.) was coated overnight with 50 ul/wellof 10 mg/ml unlabeled anti-lymphokine (IL-4, IL-10, or Interferon γ)antibody (Pharmingin San Diego, Calif.) in a 0.1 M potassium bicarbonatebuffer. The plates were washed and 100 ul I-block buffer (Tropix,Bedford, Mass.) was added for one hour. Standards (Recombinant IL-4,IL-10 and Interferon γ, Pharmingen, San Diego, Calif.) were pre dilutedin I-block beginning with a concentration of 10 ng/ml. Eight-three folddilutions of the standard were made. Samples, controls and standardswere diluted in an equal volume of I-block buffer. Aliquots of 50 ulwere added to each assay plate. The samples incubated for 1 hour at roomtemperature. The plates were washed. Secondary biotinlyated antibody wasdiluted 1:1000 in I-block and 50 ul/well was added to the assay plates.The plates were washed and 50 ul/well of avidin-alkaline phosphatase(Avidix AP, Tropix, Bedford, Mass.) was added to the assay plates. Theplates were incubated for one hour at room temperature. The washedplates were incubated twice for one minute with assay buffer (Tropix).The CDP-Star substrate (Tropix) was added to each well (100 ul/well).After 10 minutes the plates were read on a MD2250 luminometer (Dyatech,Chantilly, Va.). The first specimens were assayed using a modifiedprotocol. Instead of a detector step using avidin-alkaline phosphatase,avidin-aequorin (Sealite Sciences, Atlanta, Ga.) was used. This materialbecame unavailable during the study so the protocol was modified.Results using standard and control specimens were identical for the twoassay formats.

Serotype cross-reactivity. To examine serotype specificity, PBMCcollected on days 42, 45, or 105 from selected recipients of themonovalent attenuated vaccines (see results) were stimulated for fourdays @ 250,000 cells/well with each serotype of virus in independentcultures. Culture supernatants were then analyzed using thechemiluminescent lymphokine ELISA.

T cell subset depletions. To examine the specific cellular source oflymphokine production, PBMC were depleted of CD3+ or CD8+ T lymphocytesprior to stimulation. Selected PBMC were washed twice with RPMI medium1640 and suspended at 3.25 million cells/ml in 5% complete media (30%more PBMC were used as input to compensate for cell loss during thedepletion procedure). For the negative depletion, cells (650,000 PBMC)were incubated with washed antibody coated magnetic beads. Two types ofbeads were used, M-450 anti CD3 and anti CD8 beads (Dynal, Oslo,Norway). The anti CD3 beads were used at a concentration of 5.2 millionparticles/tube giving an approximate 20:1 bead to target cell ratio. Theanti-CD8 beads were used at a concentration of 4.0 million particles pertube giving an approximate 31:1 bead to target cell ratio.) DYNABEADS™(Dynal) in 1.5 ml microcentrifuge tubes. The cells were incubated at 4°C. for 30 minutes with moderate agitation. Non-depleted PBMC were usedas controls. Using an MPC-2 magnetic particle concentrator (Dynal)labeled cells were removed from the cell mixture. CD3+ and CD8+negatively selected PBMC were transferred to fresh microcentrifugetubes. To remove any residual unbound cells, the concentrated Dynabeadswere washed once with 200 ul complete medium. After transfer, the finalvolume (400 ul) was divided equally into two wells of a 96-well V-bottomculture plate. Depleted and control PBMC culture supernatants wereanalyzed after four days using the chemiluminescent lymphokine ELISA. Inaddition, the cultured PBMC were assayed for intracellular granzyme BmRNA (see below). CD4+ depletion was performed similarly but theseparation was done after stimulation using M-450 CD4+ (28.6 ml/4.004million particles, an approximate 31:1 bead to target cell ratio)dynabeads. CD4+ negatively selected PBMC were assayed only forintracellular granzyme B mRNA.

Flow cytometry. Depletion efficiency (measured as % depletion) wasdetermined using FACS analysis after dual staining of a randomlyselected, unstimulated PBMC population (both non-depleted control andCD3+ or CD8+ depleted sets). The cells were incubated with PE labeledanti-CD4+ or anti-CD8+ and FITC labeled anti-CD3+ antibodies(Becton-Dickinson) for 30 minutes at 4° C. Labeled PBMC were then washedthree times with fluorescence buffer [PBS (Sigma), 0.05% Na Azide, 1%Fetal Bovine Serum (Summit Biotechnology, Boulder, Colo.) and preservedin fluorescence fixative [PBS, 1% Formalin, 0.05% Na Azide] prior toanalysis. Depletion efficiency, using the CD4+ Dynabeads, was notmeasured.

Granzyme B assay. Non-depleted control PBMC and T cell subset depletedPBMC were assayed for intracellular granzyme B mRNA, after four days ofstimulation with wild-type virus. A Reverse Transcriptase PolymeraseChain Reaction (RT-PCR) assay in a 96-well plate format was used.

The mRNA purification was done using the “Straight A's” mRNA IsolationSystem (Novagen, Madison, Wis.). After centrifugation and removal ofPBMC culture supernatants for lymphokine ELISA analysis, pelleted PBMCwere lysed using 200 ul/well of lysis buffer containing 10 mMdithiothreitol and then incubated with 200 mg/well of washed oligo dTmagnetic beads for 30 minutes at room temperature. After thoroughlywashing the beads with eight volumes of wash buffer using a MPC-96(Dynal) magnetic particle concentrator to remove DNA, proteins, andcellular debris, mRNA was eluted at 70° C. for 20 minutes with 200ul/well of H₂O. The eluate was transferred to a 1.5 ml microcentrifugetube and a second round elution performed with an additional 200 ml/wellof H₂O. The 400 ul of eluate was next precipitated using 50 ul of 3Msodium acetate (pH 5.2), 20 mg of glycogen (Novagen), and 300 ul ofisopropanol. After a final wash with 70% cold ethanol, the mRNA pelletwas suspended in 30 ul of H₂O.

RT-PCR steps were performed in 96-well plates. Oligonucleotide primers(22 bp), which correspond to exons of the human granzyme B (CTLA-1) andamplify a 120 bp region, were synthesized by Dr. Stuart Cohen at theWalter Reed Army Institute of Research. The primers had the followingsequences: grb2a (sense) 5′ AGC CGA CCC AGC AGT TTA TCC C (SEQ ID NO:1),grb2b (anti-sense) 5′ C TCT GGT CCG CTT GGC CTT TCT (SEQ ID NO:2).

For each reverse transcriptase reaction, the total reaction volume was40 ul and included the following: MgCl₂ (5 mM), 10× buffer II (10 mMTris-HCL, 50 mM KCL, pH 8.3), dNTPs (1 mM each), and RNase inhibitor (40Units) [Perkin Elmer, Norwalk, Conn.] AMV reverse transcriptase (10Units) [Siekagaku], grb2b primer (3 pmoles), sH₂0, and 4 ml of mRNAtemplate. RT incubation steps were done in a 9600 thermocycler (PerkinElmer) with parameters set at 42° C. (90 minutes), 99° C. (5 minutes),4° C. (indefinitely). For each PCR, the total reaction volume was 50 uland included the following: MgCl₂ (2 mM), 10× buffer II (same as above),dNTPs (0.4 mM each), amplitaq gold (1.25 Units), grb2a and grb2b primers(1 pmole each), sH₂0, and 5 ul of cDNA template. PCR incubation stepswere also done in a 9600 thermocycler with parameters set at 95° C.initial denaturation/enzyme activation (10 minutes), 30 cycles: [95° C.denaturing (30 seconds with a 10 second ramp)/60° C. annealing (30seconds with a 30 second ramp)/72° C. extension (30 seconds with a 30second ramp)], 72° C. final extension (7 minutes), 4° C. (indefinitely).

Using electrophoresis, final amplified PCR products (10 ul) wereseparated on ethidium bromide stained 2% agarose (SeaKem)/1×TAE(Tris-Acetate-EDTA) gels and analyzed using a digital camera (ScientificImaging Systems, New Haven, Conn.).

It was reasoned that if a booster response to a booster dose of livevaccine could be demonstrated, a more attenuated live virus vaccinecould be used. The booster response sought was both an antibody and a Tcell response.

While T cell responses to dengue vaccines have been measured, fewermeasurements of T cell responses have been made than antibody responses.Therefore, the T cell response to administration of live dengue vaccineis less well characterized. One goal of this study was to determine thenature of the T cell response to the vaccines in terms of T helperresponse, serotype specificity and cytotoxic potential.

The predominating T cell response to these vaccines was a Th1 response.This was determined by the secretion of interferon γ by peripheral bloodmononuclear cells (PBMC) stimulated by live dengue virus in a four dayculture. The interferon γ was secreted by CD3+ CD8− T cells. The T cellresponse was dengue virus serotype specific with some cross-reactiveresponse. An anamnestic response was noted in some of the individualsand not others.

Lymphokine Secretion by Dengue Stimulated Cells.

Live dengue virus was used to stimulate PBMC cultures. The serotype ofstimulating virus used in culture was the same as the serotype of thevaccine virus. After four days, the tissue culture supernatants wereassayed for the presence of interferon γ, IL-4 and IL-10. In allcultures, IL-4 and IL-10 were consistently negative. Two assay controlswere used to insure that the assay was working properly. First, thestandard curve used recombinant lymphokine and second, a control samplewas used to insure that the lymphokines could be detected in thepresence of tissue culture supernatant.

In contrast to the negative expression of IL-4 and IL-10, high levels ofinterferon γ were detected in several of the culture supernatants. FIG.6 shows the kinetics of interferon γ expression of cells collected fromvolunteers receiving monovalent vaccines. Overall, the highestinterferon γ responses were by PBMC collected from recipients of dengue1 and dengue 2 candidate vaccines, though there were a few highresponses in dengue 3 and 4 recipients. The interferon γ wasoccasionally detected by the 14th day after the first inoculation butoften the expression was not detected until just prior to or just afteradministration of the second dose. The kinetics of secretion wastherefore much slower than expected. In regard to booster responses forthe monovalent recipients in this study, there were no consistentpatterns. Depending on the individual, interferon γ levels eitherincreased or decreased after administration of the second dose.

Unstimulated PBMC from all volunteers at all collection points showedundetectable levels of interferon γ. The mean expression from stimulatedday zero cells was 127 pg/ml with a standard deviation of 230 pg/ml.

For the monovalent vaccine recipients, there were 16 positive and 14negative interferon γ responders (mean±3 standard deviations). Sixteenof thirty monovalent vaccine recipients had PBMC cultures withinterferon g results >1000 pg/ml for at least one time point. Twelve hadsustained interferon g secretion at >1000 pg/ml for two or moreconsecutive time points. Also, twelve of thirty had secretion >1000pg/ml on the last time point assayed.

Four volunteers received tetravalent vaccines (an equal mixture of allfour monovalent strains). FIG. 7 shows the interferon γ production byPBMC collected from these tetravalent recipients. The PBMC werestimulated in separate cultures using one of each of the four serotypesof dengue virus. The PBMC from volunteers #33 and #36 secretedsignificant amounts of interferon γ, >1000 pg/ml, for at least one timepoint after stimulation with each of the four of the serotypes. The PBMCfrom volunteer #35 secreted significant amounts of interferon γ inresponse to three of the four serotypes (not dengue 3). The PBMC fromvolunteer #34 secreted significant interferon-gamma only in response todengue 2 virus. Highest responses were predominantly to DEN 1 and 2. Thekinetics of interferon γ production was delayed in the tetravalentvaccine volunteers as it was in the monovalent volunteers. High levelsof interferon γ were detected just prior to and after inoculation of thesecond dose. In regard to booster responses, as with the monovalentrecipients, there were no consistent interferon secretion γ patternsafter administration of the second dose.

In aggregate, these results indicate that the predominant T lymphocyteresponse in both monovalent and tetravalent vaccine recipients was anantigen specific Th1 response.

EXAMPLE 11

Serotype cross-reactivity. PBMC from twelve of the monovalent vaccinerecipients were examined for the presence of dengue serotype-specificand cross-reactive responses. Based on kinetics, those individuals whosecreted >1000 pg/ml of interferon γ in PBMC culture supernatants on thelast time point (second to last collection day) were chosen. PBMC fromthe last collection day were stimulated in independent cultures for fourdays with each dengue serotype followed by analysis of secretedinterferon γ in culture supernatants. Although there was some serotypecross-reactivity, the highest response was always seen in PBMCstimulated with the same serotype virus as the original vaccination(Table 14). Thus the interferon γ responses seen in PBMC from theseselected monovalent vaccine recipients were dengue serotype-specific.

Cross reactive responses were half (or less) of the serotype specificresponse. For Dengue 2 vaccine recipients, the highest cross-reactiveresponse was with dengue 4 virus. For dengue 4 vaccine recipients, thehighest cross-reactive response was with dengue 2 virus. For dengue 1vaccine recipients, the cross-reactive responses varied. There was onlyone dengue 3 vaccine recipient in this group and that response wasserotype specific.

Table 14. Serotype specific and cross-reactive interferon γ expressionby PBMC collected from monovalent vaccine recipients. The PBMC collectedfrom individuals receiving monovalent attenuated dengue vaccines wereseparately stimulated in culture with each of the four serotypes ofdengue virus. A subgroup of cells was selected based upon an interferonγ production of at least 1000 pg/ml in other assays. Serotype specificresponses were always the highest, however cross-reactive responses alsowere noted. Results are shown as supernatant interferon γ inpicograms/ml.

Volunteer Serotype Dengue 1 Dengue 2 Dengue 3 Dengue 4 4 1 1030 202 419129 10 1 648 58 42 73 15 1 163 0 15 0 16 1 1731 51 250 506 22 1 546 200159 47 29 1 168 0 26 0 31 1 375 0 0 0 11 2 690 5175 960 2610 20 2 7976101 850 962 3 3 0 0 714 0 12 4 1239 1987 1067 4410 13 4 445 1391 114818

EXAMPLE 12

T cell subset depletions. To verify that this was a Th1 response, theidentity of the cells secreting the interferon γ was determined. Thiswas done by depleting T cells or T cell subsets prior to culture. Thecells used in this study were mixed PBMC separated from whole bloodusing density gradient centrifugation. The predominant cells in PBMCpopulations include T cells, B cells, monocytes and NK cells. For thisassessment, we chose the time point of the highest interferon γ responsebased on kinetics in 13 monovalent and 3 tetravalent volunteers.

Cells were removed from PBMC using immunomagnetic cell separation. Thedepletion efficiency was assessed using flow cytometry in testdepletions. Analysis of the cultured PBMC was not done because of thesmall number available. In the test depletions, removal of CD3+ cellsusing CD3 monoclonal antibody resulted in a 92% reduction of CD3+ cellsrelative to non-depleted PBMC controls. The CD3 depletion was monitoredusing dual labels for CD3 and CD4, dual labels for CD3 and CD8, andsingle label for CD3. The CD3 depletion was more thorough for CD4+ cellsthan CD8+ cells with 98% of the CD3/CD4 T cells being depleted and 90%of the CD3/CD8 cells being depleted in the CD3 depleted groups. Removalof CD8+ cells using CD8 monoclonal antibody resulted in a 99.9%reduction of CD8+ cells.

Selected PBMC were depleted of CD3+ or CD8+ T lymphocytes, stimulated inculture with dengue virus for four days, and then examined for secretedinterferon γ. Results were compared to those obtained from non-depletedPBMC controls cultured at the same time. CD4+ T lymphocytes were notdepleted prior to stimulation because other cell populations need CD4+ Thelp for production of interferon γ.

Removal of CD3+ cells prior to culture substantially reduced theproduction of interferon γas shown in Table 15. The range for reductionin interferon γ after CD3+ depletion was 59–100%. Reduced butsignificant interferon γ production was seen in some CD3+ depletedcultures. This residual production indicates that either the smallamount of residual CD3+ cells remaining after immunomagnetic cellseparation are secreting interferon γ and/or another population of cellsis also secreting interferon γ.

Table 15. Lymphocytes secreting (or inducing the secretion of)interferon γ are CD3+ CD8-T cells. Selected lymphocyte subsets werenegatively depleted using immunomagnetic cell separation techniques. Theremaining cells were stimulated with live dengue virus for four days andthe culture supernatant was assayed for interferon γ. Depletion of CD3+lymphocytes prior to culture negatively influenced the production ofinterferon γ.

Control CD3 depleted CD8 depleted Interferon Interferon InterferonVolunteer γ (pg/ml) γ % Change γ % Change  3 883 0 100 565 36  4 30841038 66 5559 [80] 10 4295 1781 59 4271   0.6 11 525 88 83 633 [21] 1210000 1017 90 10000  0 13 5977 385 94 8392 [40] 15 1365 255 81 1910 [40]16 1861 84 95 2113 [14] 17 576 42 93 1265 [120]  20 4614 1329 71 4235  822 1303 39 89 1349 [3.5]  29 2478 5 99 5681 [129]  31 995 370 63 3057[207]  33T 2393 9 99 2114 12 35T 10000 202 98 9637  4 36T 3542 469 873257  8

Except in one individual, removal of CD8+ cells prior to culture did notreduce the production of interferon γ. In 9 of the 16 cultures, removalof CD8+ cells actually increased its production, possibly due to removalof suppression by these cells or by reducing the killing of infectedantigen presenting cells by CD8+ cytotoxic lymphocytes.

Together, these results indicate that the interferon γ seen in thesePBMC cultures is either secreted by CD4+ T lymphocytes and/or by cellsinfluenced by CD4+ T lymphocytes. This supports the finding of a Th1response.

EXAMPLE 13

Granzyme B. A Th1 response is associated with, among other things, acytotoxic lymphocyte response. In an effort to see if cells capable ofcell mediated killing were present in these vaccine volunteers, granzymeB mRNA was measured in the PBMC cultured for the depletion experiments.After removal of culture supernatants for lymphokine analysis, the cellswere pelleted and lysed for extraction of mRNA. Granzyme B specificprimers were used for RT-PCR. The PCR product was analyzed by agarosegel electrophoresis. Gel band intensity was converted into a +, − scaleusing a reference photograph (FIG. 8) for comparison. Extra cells fromseven of the volunteers were cultured without virus. The unstimulatedPBMC, from these seven volunteers, had little (− or +) granzyme B mRNAexpression. With antigen-specific stimulation, expression wassubstantially upregulated in all 16 of the selected vaccine recipients(FIG. 8). T cell subset depletion using CD8 monoclonal antibody did notsignificantly reduce granzyme B expression relative to control PBMC.There were 3 individuals (ID 16, 22, and 33) whose granzyme B expressionwas reduced in the CD8 depleted group. In one (ID 33), the decrease wassubstantial. In contrast, T cell subset depletion using CD3 monoclonalantibody reduced expression in 14 of the volunteers. In 8 of themonovalent volunteers and in all 3 tetravalent volunteers, the decreasewas substantial. Four of the interferon γ non-responders were alsoexamined for granzyme B mRNA. All showed low levels of expression (datanot shown).

In cells from seven of the volunteers, T cell subset depletion using CD4monoclonal antibody was done after the four days of culture. Thedepletion was done after culture in order to provide T helper activityto all cells needing help during culture. Removal of CD4+ cells afterstimulation did not affect granzyme B expression relative tonon-depleted controls in the seven volunteers analyzed. Thus, althoughthere is an antigen dependent production of granzyme B mediated by CD4+Th1 cells, the actual cells that produce the granzyme B appear to becells other than T cells. Whether this is production by NK cells ormacrophages is unknown.

Discussion

Two objectives of this study were to determine if there was a measurableT cell response in the vaccine recipients and if a cell mediatedresponse to the second dose of vaccine could be seen. For thoseobjectives, T cell response kinetics were measured by re-stimulatingcells collected at intervals around the two doses. The re-stimulationwas done with live virus in bulk cultures of PBMC collected during thestudy.

A third objective of this study was to determine the nature of the Tcell response in terms of 1. cell type defined by lymphokine repertoire,2. dengue serotype specific and cross-reactive responses, and 3. ameasure of cytotoxic potential, granzyme B production. These responseswere measured in PBMC from both monovalent and tetravalent vaccinerecipients. In regard to the tetravalent vaccine recipients, it wasimportant to determine if a response could be detected to all fourserotypes of dengue virus.

Human and mouse T helper responses can be divided into two groups basedupon their pattern of lymphokine expression 5. T helper 1 (Th1) cellsare characterized by the secretion of IL-2 and interferon γ. Of thosetwo lymphokines, interferon γ is the most important in terms ofidentifying Th1 cells. T helper 2 (Th2) cells are characterized by thesecretion of IL-4, IL-5, IL-6 and IL-10. In mixed populations of cellsor PBMC bulk culture, one of the two secretion patterns usuallypredominates.

One factor influencing the Th1 vs Th2 response is the nature of theassaulting infection. Viral infections, and some bacterial infectionssuch as Listeria and Mycobacterium (Peters, 1996, Hepatology 23,909–916) often induce a Th1 response while some parasitic infectionswill induce a Th2 response (Conrad et al., 1990, J Exp Med 171,1497–1508). The proportion of the two responses may vary during thecourse of the infection. For instance, even though viral infectionsusually beg in with a Th1 response, a Th2 response can be produced laterin the infection. The initial Th1 response may augment CTL responses anddirect immunoglobulin isotype switching while the following Th2 responsemay augment antibody production by B cells.

In natural dengue infection, one study showed a Th1 response in mostindividuals. The Th1 response was associated with an effective immuneresponse without associated severe pathogenesis. In contrast, someindividuals developed a Th2 response that was associated with greaterpathogenesis.

In spite of the association of a Th1 response with an effectiveanti-dengue immune response, the key lymphokine of a Th1 response,interferon γ, has both positive and negative influences on the immuneresponse. In Thailand, Kurane found high levels of interferon γ in theserum of DHF patients in comparison to lower levels in the serum of DFpatients (Kurane et al., 1991, J Clin Invest 88, 1473–1480). Theincreased interferon γ may be a measure of immune activation. Interferonγ is needed to activate and maintain activation of cytotoxic cells (CD4+T cells, CD8+ T cells and NK cells). While this mechanism may contributeto pathogenesis in severe infections, the same response may bebeneficial in milder infections by reducing the number of virallyinfected cells through antigen specific cytolysis. The positive role ofinterferon γ in controlling dengue virus infection is demonstrated in arecent mouse knockout model deficient in interferons α, β and γ. Theknock-out mice were susceptible to lethal infection by dengue viruses incontrast to normal adult controls that were resistant to infection(Johnson and Roehrig, 1999, J Virol 73, 783–786).

Alternatively, interferon γ may contribute to the pathogenesis of denguevirus infection. One mechanism for the pathogenesis may be by immuneenhancement due to increasing the infection of one major target cell,the macrophage. In culture, interferon γ increased the antibody-mediatedinfection of a macrophage cell line U937 by increasing the number of Fcreceptors on the surface of the cells (Kontny et al., 1988, J Virol 62,3928–3933). Although another study using normal cultured macrophagesshowed the opposite effect of decreasing the infection (Sittisombut etal., 1995, J Med Virol 45, 43–49). Given these conflicting results, itis unclear whether interferon γ contributes to increased infection ofmacrophages.

In this study, a Th1 response was the predominant response. Assays forIL-4 and IL-10 were consistently negative indicating a lack of TH2response. High levels of interferon γ were detected in the supernatantsof many of the cultures, indicating the presence of a Th1 response inthose cultures.

Since the stimulated cells were whole PBMC, the cells responsible forsecretion of the interferon γ needed to be determined. This was done bydepleting T cell subsets using an immunomagnetic procedure. Negativedepletion was done prior to culture with antibodies recognizing eitherCD3 or CD8. Since CD3 depletion resulted in abrogation of interferon γsecretion and CD8 depletion did not, it was concluded that CD3+ CD8−lymphocytes were the cell population secreting the interferon γ or atleast controlling the secretion of interferon γ. This confirms that theinterferon γ was the result of a Th1 response. Residual interferon γ insome cultures after depletion may have been due to some remaining CD4+ Tlymphocytes after depletion or other cells in the culture, possiblyNatural Killer cells or macrophages.

The peak interferon γ response was serotype specific. When cells frommonovalent vaccine recipients were stimulated separately by each of thefour serotypes of dengue viruses, the peak interferon γ production wasin response to stimulation by dengue virus homologous to the vaccinevirus. Lesser, cross-reactive responses to other dengue viruses werenoted in several of the cultures. This is similar to the resultsobtained by others using a different measurement, lymphocyteproliferation. In one study, cells from individuals receiving a dengue 2vaccine exhibited the greatest response to dengue 2 virus butcross-reactive responses were noted (Dharakul, J Infect Dis 170, 27–33).This was confirmed at the clonal level where the majority of clonesobtained from a dengue 3 vaccine recipient responded best to dengue 3antigen but had cross reactive responses to the other three dengueantigens (Kurane et al., 1989, J Exp Med 170, 763–775). The conclusionof the latter study was that primary dengue virus infection producespredominantly cross-reactive CD4+ lymphocyte responses (proliferationand interferon γ production).

In this study, cross-reactive responses of monovalent vaccinerecipients' PBMC were usually half or less of the serotype specificresponse. In the tetravalent vaccine recipients, interferon γ secretionin response to individual serotypes of dengue virus was significant inthree out of four tetravalent vaccine recipients. The responses variedwithin individual vaccine recipients enough that it was not possible todetermine if the lower responses were serotype specific responses orcross-reactive responses.

The kinetics of T cell activation as indicated by interferon γ secretionwas slower than expected. In a few instances, responses could bedetected by day 14. However in most cases, responses were not detecteduntil just prior to administration of the second vaccine dose. It isunclear what the reason is for the delayed kinetics. One explanationcould be that antigen production by vaccine virus infected cells is slowand persistent. However, it is equally possible that the methodspreferentially detected memory responses rather than acute responses.For instance, if active CD8+ cells were inhibiting a CD4+ response inPBMC collected during early infection, a measurable response may beattenuated. In cultures where the CD8+ lymphocytes were depleted,interferon γ secretion by the remaining lymphocytes was increased inmore than half of the cultures. This inhibition may have been greaterduring early infection.

Others have observed more acute lymphokine production kinetics. Serumlymphokines, including serum interferon γ were measured for 17 daysafter inoculation with an attenuated dengue vaccine. An acute responsewas noted in that study that peaked during the time of viremia (Kuraneet al., 1995, J Clin Lab Immunol 46, 35–40).

The response to the second dose was mixed. Some individuals showed anincrease in interferon γ production while others showed a decrease. Theinterferon γ production by cells collected from vaccine recipients justprior to the second dose was high enough that it may have masked anyanamnestic response to the second dose. In addition, the late interferonγ response may have made the measurement of an anamnestic T cellresponse more difficult. It is clear that some individuals responded tothe second dose. This may indicate that there is some localized virusgrowth in the presence of an active immune response.

In summary, the predominant T cell response to administration of theselive attenuated dengue viruses was a Th1 response. This was demonstratedby the secretion of interferon γ by re-stimulated PBMC collected fromvaccine recipients. None of the PBMC cultures from vaccine recipient'scells had significant IL-4 or IL-10 secretion into the culturesupernatant after re-stimulation. The Th1 response was verified byshowing that CD3+ CD8− lymphocytes were secreting the interferon γ. TheTh1 response was predominantly dengue serotype specific but smallercross-reactive responses were noted.

EXAMPLE 14

Clinical and Immunological Evaluations of Four Dengue Viruses asChallenge Strains in Immune and Susceptible Volunteers.

The primary objective of this study is to characterize clinicalresponses to each of 4 candidate dengue challenge viruses in susceptibleand immune volunteers to judge their suitability as challenge strainsfor human vaccine efficacy studies. The secondary objective of the studyis to generate hypotheses regarding the immune correlates of protectionfor dengue fever.

Dose, Schedule and Route: All volunteers will receive either 1 of 4dengue challenge viruses or placebo in a single dose of 0.5 mlsubcutaneously in the deltoid region on study day 0.

Study Groups:

Volunteer Set #1 (susceptible): to receive either DEN-1, DEN-2, DEN-3,DEN-4 or placebo Volunteer Set #2 (immune): to receive either DEN virus(serotype corresponding to previously received vaccine) or placebo

General Eligibility Criteria: Age 18–35, excellent health without anychronic medical conditions, score of >75% on written study-comprehensionexamination, informed consent, availability for the study period, letterof approval for participation from chain of command (military only),serologic conversion in response to previous dengue vaccination(volunteer set #2 only)

Statistics: Data analysis will be primarily descriptive in this pilotstudy given the small number of volunteers in each test article group.The primary concern will be to document the frequency of clinical events(pre-specified and unexpected) within the four study groups as comparedto placebo.

Pre-challenge immune measures and post-challenge immune responses of allchallenged volunteers who develop dengue fever will be compared to thoseof all challenged volunteers who remain well, to develop hypothesesabout immune correlates of protection.

Application of the Human Dengue Challenge Model: In contrast to mosthistorical human dengue challenge experiments which were designed eitherto characterize dengue illness or to evaluate the attenuation of livevaccine candidates, this challenge study will aim to 1) validate 4dengue viruses as challenge strains in flavivirus-naïve volunteers(volunteer set #1), and 2) identify correlates of immunity in recipientsof monotypic dengue vaccine when subsequently challenged with homotypicdengue virus (volunteer set #2).

If the clinical response in volunteer set #1 suggests these strains aresuitable for challenge, then in future controlled experiments, thesechallenge strains will be administered to recipients of dengue vaccinecandidates or placebo to select the most promising vaccine candidatesfor further development.

If the immunological response in volunteer set #2 suggests that someaspect of pre-challenge immunity (antibodies and/or T cell memory)correlate with protection, such correlates of protection could simplifydengue vaccine development.

Defining Criteria for Dengue Viruses to be Tested as Challenge Strains:A suitable dengue challenge virus will 1) reproducibly causeuncomplicated dengue fever lasting 3–7 days in volunteer set #1, 2) beproduced in compliance with Good Manufacturing Practices (GMP) and befree of adventitious agents or reactogenic non-viral components, and 3)be available as lyophilized virus in sufficient quantity (>100 doses).Challenge Viruses include DEN-1 45AZ5 (PDK-0) inactivated, DEN-2 S16803(PDK-10), DEN-3 cl 24/28 (PDK-0), DEN-4 341750 (PDK-6) (PDK=primary dogkidney cells).

Dose of each challenge virus in plaque forming units (pfu.) will be 0.5×titer.

The study challenge viruses meet the latter two criteria. This studyaims to demonstrate that the study candidate challenge strains meet thefirst criterion. We have some evidence that the 4 challenge viruses tobe tested in this study are appropriately pathogenic. The DEN-1 andDEN-3 challenge viruses have already been shown to cause uncomplicatedfebrile illness in volunteers. Though the DEN-2 and DEN-4 challengeviruses to be administered in this study are untested in volunteers,they are believed to be pathogenic, as they are the precursors of denguevirus vaccine candidates that were rejected because they caused febrileillnesses in volunteers. The only reason for rejecting any of the 4study candidate dengue challenge viruses is if they cause either noillness in flavivirus-naïve volunteers (volunteer set #1) or excessiveillness in any volunteer (volunteer sets #1 and #2).

Volunteer Set #1: Ten healthy flavivirus-naïve volunteers will berandomized to receive dengue virus challenge with 1 of 4 serotypes (2volunteers per serotype) or placebo. The volunteers and investigatorswill remain blinded to the inoculum. We expect that the 8 volunteers whoreceive dengue challenge viruses will become moderately ill with 3–7days of fever, severe headache and myalgias. Full recovery may take aslong as 14 days from onset of illness. Each challenge virus will bedeemed suitable based on the clinical responses of the 2 recipients andmust satisfy the study definition of dengue fever. Dengue fever isdefined as: an illness with: 2 or more of the following: headache,myalgia, erythematous rash, retro-orbital pain, arthralgias, andsustained fever for 48 hours or more allowing for periods of decreasedtemperature due to acetaminophen use, and tissue response during periodof fever and days thereafter manifest by neutropenia or thrombocytopeniaor liver injury, and evidence of dengue viremia during the period offever.

Volunteer Set #2: Up to twelve young, healthy immune volunteers willreceive homotypic dengue challenge virus (N=10, regardless of serotype)or placebo (N=2). Immune volunteers are previous recipients ofmonovalent, live-attenuated dengue vaccines who had a primaryneutralizing antibody response. Immune volunteers are expected to remainwell. Measures of their pre-challenge immune status or immune activationintra-challenge may identify correlates of protection.

1. A method for stimulating dengue virus specific immune response, whichcomprises administering to an individual an immunologically sufficientamount of two or more attenuated viruses chosen from the groupconsisting of a dengue-1 (DEN-1) virus having the sequence of DEN-1strain 45AZ5 PDK-27 having the ATCC accession number PTA-4810, adengue-2 (DEN-2) virus having the sequence of DEN-2 strain S16803 PDK-50having the ATCC accession number VR-2653, a dengue-3 (DEN-3) virushaving the sequence of DEN-3 strain CH53489 PDK-20 having the ATCCaccession number VR-2647, and a dengue-4 (DEN-4) virus having thesequence of DEN-4 strain 341750 PDK-6 having the ATCC accession numberPTA-4811, and a physiologically acceptable vehicle.
 2. The method ofclaim 1, wherein the attenuated virus is administered parenterally. 3.The method of claim 1, wherein the attenuated virus is administeredintranasally.
 4. The method of claim 1, which comprises administering toan individual an immunologically sufficient amount of a dengue-1 (DEN-1)virus having the sequence of DEN-1 strain 45AZ5 PDK-27 having the ATCCaccession number PTA-4810, a dengue-2 (DEN-2) virus having the sequenceof DEN-2 strain S16803 PDK-50 having the ATCC accession number VR-2653,a dengue-3 (DEN-3) virus having the sequence of DEN-3 strain CH53489PDK-20 having the ATCC accession number VR-2647, and a dengue-4 (DEN-4)virus having the sequence of DEN-4 strain 341750 PDK-6 having the ATCCaccession number PTA-4811, and a physiologically acceptable vehicle. 5.The method of claim 1, which further comprises administering an adjuvantto enhance the immune response.
 6. The method of claim 1, wherein theattenuated viruses administered are formulated in a dose of 10² to 10⁶PFU/ml.
 7. The method of claim 1, wherein the attenuated viruses areadministered subcutaneously.
 8. A method for stimulating dengue virusspecific immune response, which comprises administering to an individualan immunologically sufficient amount of two or more attenuated viruseschosen from the group consisting of a dengue-1 (DEN-1) virus having thesequence of DEN-1 strain 45AZ5 PDK-27 having the ATCC accession numberPTA-4810, a dengue-2 (DEN-2) virus having the sequence of DEN-2 strainS16803 PDK-50 having the ATCC accession number VR-2653, a dengue-3(DEN-3) virus having the sequence of DEN-3 strain CH53489 PDK-20 havingthe ATCC accession number VR-2647, and a dengue-4 (DEN-4) virus havingthe sequence of DEN-4 strain 341750 PDK-20 having the ATCC accessionnumber VR-2652, and a physiologically acceptable vehicle.
 9. The methodof claim 8, which comprises administering to an individual animmunologically sufficient amount of a dengue-1 (DEN-1) virus having thesequence of DEN-1 strain 45AZ5 PDK-27 having the ATCC accession numberPTA-4810, a dengue-2 (DEN-2) virus having the sequence of DEN-2 strainS16803 PDK-50 having the ATCC accession number VR-2653, a dengue-3(DEN-3) virus having the sequence of DEN-3 strain CH53489 PDK-20 havingthe ATCC accession number VR-2647, and a dengue-4 (DEN-4) virus havingthe sequence of DEN-4 strain 341750 PDK-20 having the ATCC accessionnumber VR-2652, and a physiologically acceptable vehicle.
 10. A methodfor stimulating dengue virus specific immune response, which comprisesadministering to an individual an immunologically sufficient amount oftwo or more attenuated viruses chosen from the group consisting of adengue-1 (DEN-1) virus having the sequence of DEN-1 strain 45AZ5 PDK-20having the ATCC accession number VR-2648, a dengue-2 (DEN-2) virushaving the sequence of DEN-2 strain S16803 PDK-50 having the ATCCaccession number VR-2653, a dengue-3 (DEN-3) virus having the sequenceof DEN-3 strain CH53489 PDK-20 having the ATCC accession number VR-2647,and a dengue-4 (DEN-4) virus having the sequence of DEN-4 strain 341750PDK-20 having the ATCC accession number VR-2652, and a physiologicallyacceptable vehicle.
 11. The method of claim 10, which comprisesadministering to an individual an immunologically sufficient amount of adengue-1 (DEN-1) virus having the sequence of DEN-1 strain 45AZ5 PDK-20having the ATCC accession number VR-2648, a dengue-2 (DEN-2) virushaving the sequence of DEN-2 strain S16803 PDK-50 having the ATCCaccession number VR-2653, a dengue-3 (DEN-3) virus having the sequenceof DEN-3 strain CH53489 PDK-20 having the ATCC accession number VR-2647,and a dengue-4 (DEN-4) virus having the sequence of DEN-4 strain 341750PDK-20 having the ATCC accession number VR-2652, and a physiologicallyacceptable vehicle.
 12. A method for stimulating dengue virus specificimmune response, which comprises administering to an individual animmunologically sufficient amount of two or more attenuated viruseschosen from the group consisting of a dengue-1 (DEN-1) virus having thesequence of DEN-1 strain 45AZ5 PDK-20 having the ATCC accession numberVR-2648, a dengue-2 (DEN-2) virus having the sequence of DEN-2 strainS16803 PDK-50 having the ATCC accession number VR-2653, a dengue-3(DEN-3) virus having the sequence of DEN-3 strain CH53489 PDK-20 havingthe ATCC accession number VR-2647, and a dengue-4 (DEN-4) virus havingthe sequence of DEN-4 strain 341750 PDK-6 having the ATCC accessionnumber PTA-4811, and a physiologically acceptable vehicle.
 13. Themethod of claim 12, which comprises administering to an individual animmunologically sufficient amount of a dengue-1 (DEN-1) virus having thesequence of DEN-1 strain 45AZ5 PDK-20 having the ATCC accession numberVR-2648, a dengue-2 (DEN-2) virus having the sequence of DEN-2 strainS16803 PDK-50 having the ATCC accession number VR-2653, a dengue-3(DEN-3) virus having the sequence of DEN-3 strain CH53489 PDK-20 havingthe ATCC accession number VR-2647, and a dengue-4 (DEN-4) virus havingthe sequence of DEN-4 strain 341750 PDK-6 having the ATCC accessionnumber PTA-4811, and a physiologically acceptable vehicle.